the sweet tooth of bacteria: common themes in …dance, importance, and niche speciﬁcity of the...

The Sweet Tooth of Bacteria: Common Themes in BacterialGlycoconjugates

Hanne L. P. Tytgat,a,b Sarah Lebeera,b

University of Antwerp, Department of Bioscience Engineering, Antwerp, Belgiuma; KU Leuven, Centre of Microbial and Plant Genetics, Leuven, Belgiumb

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .373GENERAL THEMES IN GLYCOCONJUGATE BIOSYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .373

Glycosyltransferases, the Key Enzymes of Glycosylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .373Sequential Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375En Bloc Glycosylation of Glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375

BACTERIAL GLYCOCONJUGATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376Peptidoglycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376Cell Wall Polysaccharides: Capsular Polysaccharides and Exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .378Macroamphiphilic Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .380

Lipopolysaccharides and lipooligosaccharides in Gram-negative bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .380Lipoglycans in Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .383Glycosylated teichoic acids in Firmicutes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .384

Protein Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .385En bloc N-glycosylation of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .386Sequential N-glycosylation of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .389Dedicated O-glycosylation of proteins: flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .389Dedicated O-glycosylation of proteins: pili . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .391Dedicated O-glycosylation of other proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .392General O-glycosylation systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394

(i) General O-glycosylation of neisserial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394(ii) General protein O-glycosylation in Bacteroidetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .396(iii) Francisella tularensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .397(iv) Acinetobacter baumannii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .398(v) Peculiar glycoproteins in Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .398(vi) Indications of general O-glycosylation systems in other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399

S-glycosylation of proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399S-layer proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399

COMMONALITIES IN GLYCOCONJUGATE BIOSYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399General Themes in Glycoconjugate Biosynthesis Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400

The lipid carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Priming or initiating GTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Transport of the lipid-linked oligosaccharide across the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Glycan ligation to the substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Common themes facilitate new discoveries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400

Overlap in Glycoconjugate Biosynthesis Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Bifunctional and Promiscuous GTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402

SUMMARY

Humans have been increasingly recognized as being superorgan-isms, living in close contact with a microbiota on all their mucosalsurfaces. However, most studies on the human microbiota havefocused on gaining comprehensive insights into the compositionof the microbiota under different health conditions (e.g., entero-types), while there is also a need for detailed knowledge of thedifferent molecules that mediate interactions with the host. Gly-coconjugates are an interesting class of molecules for detailedstudies, as they form a strain-specific barcode on the surface ofbacteria, mediating specific interactions with the host. Strikingly,most glycoconjugates are synthesized by similar biosynthesismechanisms. Bacteria can produce their major glycoconjugates by

using a sequential or an en bloc mechanism, with both mechanisticoptions coexisting in many species for different macromolecules.In this review, these common themes are conceptualized and il-lustrated for all major classes of known bacterial glycoconjugates,with a special focus on the rather recently emergent field of glyco-sylated proteins. We describe the biosynthesis and importance ofglycoconjugates in both pathogenic and beneficial bacteria and in

Address correspondence to Sarah Lebeer, [email protected].

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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both Gram-positive and -negative organisms. The focus lies onmicroorganisms important for human physiology. In addition,the potential for a better knowledge of bacterial glycoconjugates inthe emerging field of glycoengineering and other perspectives isdiscussed.

INTRODUCTION

During the last decades, it became clear that humans and, byextension, eukaryotes in general carry a heavy bacterial load.

Our own body cells are in fact outnumbered by the prokaryotic cellspresent. All human mucosal surfaces of healthy individuals, i.e., theoronasopharyngeal cavity, urogenital tract, gastrointestinal tract(GIT), upper respiratory tract, and skin, are covered with bacteria.This microbiota plays important roles in the physiology of the host;the most important and best known are the roles in the digestion ofnutrients and protection of the host against pathogens (1).

Pathogenic and beneficial members of the microbiota establishan intimate interaction with the host mucosa in order to manip-ulate the host metabolism and immune system (2). Keys to a betterunderstanding of these interactions are molecules present on thebacterial cell surface and secreted into the environment. Often,these key molecules are glycoconjugates such as glycoproteins,exopolysaccharides (EPSs), capsular polysaccharides (CPSs),lipopolysaccharides (LPSs), lipooligosaccharides (LOSs), lipogly-cans, peptidoglycan (PG), glycosylated teichoic acids (TAs), andother glycosylated secondary cell wall polymers. The glycans pres-ent on these molecules show an enormous diversity in monosac-charide building blocks, anomeric configuration, conformation,and stereochemistry (3), which largely exceeds the eukaryotic gly-coconjugate repertoire. The resulting diversity is uncanny: for in-stance, two glucose residues can already be joined together in 30different ways (4). Bacteria are also able to produce “exotic” raresugars like bacillosamine (Bac), present on glycoproteins of Cam-pylobacter jejuni (5), in contrast to the 10 monosaccharides thatare typically detected in mammals (3). The prominent location ofbacterial glycoconjugates on the cell wall and their enormous di-versity suggest that they form a unique barcode on bacterial cellsurfaces. This makes them ideal candidates to establish specificand “tight” interactions with host cells and abiotic surfaces, rang-ing from adhesion to immunomodulation (6). Of particular in-terest are various lectin immune receptors with different specific-ities displayed by host cells to scan these bacterial barcodes andinduce specific responses (7). This is crucial in view of the abun-dance, importance, and niche specificity of the microbiota andpathogenic infections. Most studies of the microbiota focus onmapping the microbiota and microbiome under different healthconditions (8–10), with work on enterotypes being some of themost widely discussed findings of this research (11). These generalstudies need to be complemented with dedicated studies on thebacterial mediators of specific interactions, such as glycoconju-gates, to generate a comprehensive view of our bacterial friendsand foes. Currently, studies of glycoconjugates in pathogenslargely outnumber those of glycoconjugates in beneficial bacteria.This discrepancy is especially apparent for glycoproteins. More-over, the field of (bacterial) glycobiology is enigmatic: an under-standing of the ties between glycan structures and their biologicalfunction is hampered by the nontemplate nature of glycan biosyn-thesis and the resulting heterogeneity. In addition, their enor-mous diversity and versatility make their study quite challenging(12). Considering the large amount of energy that cells dedicate to

the buildup of glycans, their functional importance, from an evo-lutionary perspective, should be high. Also, a better fundamentalknowledge of bacterial glycomes can open up new horizons in thediscovery of new drugs, bioactive compounds, and vaccines. Acombination of existing and newly emerging technologies is rap-idly advancing the field of glycobiology. This review aims at givingan overview of the current knowledge on bacterial glycoconju-gates and focuses on the commonalities of their biosynthesismechanisms in both Gram-positive and -negative species andfunctions in bacterium-host interactions in both pathogens andbeneficial bacteria.

GENERAL THEMES IN GLYCOCONJUGATE BIOSYNTHESIS

Strikingly, bacteria use two main pathways to synthesize a pleth-ora of glycoconjugates. The two main modular mechanisms usedfor glycoconjugate biosynthesis are the sequential and en blocmechanisms. This review conceptualizes and illustrates these twobiosynthesis mechanisms. The commonality of these mechanismsis also illustrated by the promiscuity of some glycosyltransferases(GTs), which are able to glycosylate different substrates in somebacterial species. For instance, in several pathogens, GTs are activein both LPS/LOS and glycoprotein biosynthesis pathways (13, 14).More specific examples of these commonalities are discussedbelow.

Glycosyltransferases, the Key Enzymes of Glycosylation

Glycoconjugates do not fit into a linear understanding of the flowof biological information, as the glycome is not a template-drivenprocess such as DNA, RNA, and protein biosynthesis. In contrast,the glycan structures that are present on glycoconjugates are de-termined primarily by the complement of glycan-modifying en-zymes present in an organism (12). GTs, glycoside hydrolases orglycosidases, transporters, flippases, polymerases, and other en-zymes are all required to synthesize and metabolize the glycome ofan organism. The crucial enzymes in the buildup of glycans are theGTs, which is underlined by their presence in the genome: GTsand glycosidases are believed to constitute 1 to 3% of the geneproducts of bacterial, eukaryotic, and archaeal organisms (15).This percentage is higher in plants and bacterial members of themicrobiota such as Bacteroides and Bifidobacterium, which sug-gests an important role of glycans in interactions with the humanhost (15, 16). Parasitic and symbiotic life forms, on the otherhand, generally contain fewer glycosylation enzymes (15). It re-mains to be seen if this figure needs to be updated as the fields of(meta)genomics and glycomics further evolve.

GTs (EC 2.4.x.y.) sensu stricto catalyze the formation of glyco-sidic bonds between a sugar moiety from an activated donor mol-ecule and a specific substrate molecule. GTs can use a range ofdonor molecules, both nucleotide and nonnucleotide ones, andcan target a wide diversity of substrates, such as saccharides, pro-teins, nucleic acids, proteins, lipids, and small molecules (17).Chemically, these enzymes catalyze the transfer of a glycosyl groupto a nucleophilic group, usually an alcohol. The net result of thisreaction is an O-, N-, S-, or C-glycosylated acceptor molecule (17).The glycosidic bond is established by retainment (e.g., UDP-glu-cose turns into an �-glucoside) or inversion of the stereochemis-try of the anomeric carbon of the donor sugar monomer (whichresults in a �-glucoside for UDP-glucose). These two stereochem-ical outcomes of reactions catalyzed by GTs are also used as ameans to classify these enzymes.

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Inverting GTs use a direct-displacement SN2-like reaction tocatalyze the formation of the glycosidic bond (18). The result is anet inversion of the stereochemistry of the anomeric reaction cen-ter of the donor substrate. Most of the inverting GTs require adivalent cation, typically Mg2� or Mn2�, which plays the role of anacid catalyst, coordinated by the nonstringent D-X-D motif, al-though metal-independent GTs have also been reported (17, 19).For instance, SpsA, a GT involved in the biosynthesis of the sporecoat of Bacillus subtilis, was one of the first bacterial inverting GTsto be characterized (20, 21).

The catalytic mechanism used by retaining GTs is still debated,and two hypothetical mechanisms are described in the literature.Both mechanisms can require a divalent cation coordinated by theD-X-D motif for their catalytic activity. The first mechanism wasextrapolated from knowledge on retaining glycosidases (the elu-cidation of the inverting GT mechanism), which means that re-taining GTs would use a double-displacement mechanism with ashort-lived glycosyl-enzyme intermediate (22). However, despitenumerous efforts, no such glycosyl-enzyme intermediate could betrapped (23). The second proposed mechanism suggests a directattack of the acceptor by an SNi-like or “internal return” mecha-nism (24). This hypothesis originated from research on the crystalstructure of the LOS galactosyltransferase LgtC of Neisseria men-ingitidis (25). Recently, this SNi-like mechanism was further sup-ported by mechanistic evidence, which makes the latter mecha-nism more plausible (26).

GTs can also be classified according to the donor substrates thatthey recognize. GTs that use sugar mono- or diphosphonucle-otides (e.g., UPD-Glc and GDP-Man) are termed Leloir GTs, inhonor of Nobel prize winner Leloir, who performed groundbreak-ing work on sugar nucleotides (27). GTs utilizing nonnucleotidedonors, such as polyprenol pyrophosphate and sugar-1-phos-phate, are then called non-Leloir GTs (17).

The biochemical characterization of GTs has proven to be dif-ficult, as the first reports of the X-ray crystal structures of GTsstarted to emerge only in the late 1990s (28). Although the simi-larities between glycosidases and GTs are quite high, GTs appearto exhibit only a narrow spectrum of folds. The Leloir enzymes canbe split into two large families based on their common three-dimensional (3D) fold: the GT-A and GT-B fold families (17).More recently, a third fold category was discriminated, the GT-Cfold (29). An important caveat here is that not all enzymes thatrepresent a GT-A or GT-B fold are GTs. Also, non-Leloir GTspossess non-GT-A and non-GT-B folds. An example of such GTsis the transglycosylase penicillin-binding protein 2 (PBP2) ofStaphylococcus aureus involved in PG biosynthesis, which has abacteriophage-lysozyme-like fold (30).

As GTs are a very diverse group of enzymes, and they can rec-ognize a range of donor and acceptor molecules, a classificationwas proposed by the group of Henrissat based on the amino acidsequence of GTs, analogous to the classification of glycosidehydrolases (31). These constantly updated GT families can be con-sulted online in the Carbohydrate-Active enZymes (CAZy) data-base (http://www.cazy.org/) (32). By using algorithmic methodsto subdivide protein sequences into distinct families, GTs are cur-rently classified into 96 families (July 2014). Each GT family con-tains enzymes related by sequence and 3D fold. As the mechanismof catalysis, and additionally their inverting or retaining character,is (largely) conserved within each family, this classification can beused to predict the mode of action of newly predicted GTs. Nev-

ertheless, this does not mean that all enzymes of a family recognizethe same donor and/or acceptor: polyspecificity is commonamong GT families (33). One should be prudent with the over-interpretation of predictions based purely on the classification of aGT in a certain family (34).

A few “special classes of GTs” deserve some extra focus, i.e., thetransglycosylases of PG biosynthesis pathways, synthases catalyz-ing EPS/CPS formation, and enzymes of the en bloc glycosylationpathway, such as priming GTs, polymerases, and oligosaccharyl-transferases (OSTs).

Transglycosylases are GTs involved in the polymerization ofcarbohydrate chains to PG. These enzymes follow the same mech-anism as that of retaining glycoside hydrolases, and their actioncan be described by a nucleophilic substitution reaction, resultingin an inversion of the configuration (35). Transglycosylases uselipid II (see below) as an acceptor and add these new PG subunitsprocessively to the nonreducing end of the growing chain, which isthe donor (36–38). Transglycosylases are classified into CAZyfamily 51 and have a lysozyme-like fold (30). These enzymes occuras monofunctional or bifunctional enzymes; the latter combinetheir GT action with a transpeptidase domain (39).

Synthases are multifunctional processive GTs that catalyze theinitiation, polymerization, and transport of usually rather simpleglycans. These enzymes are involved in the biosynthesis of, e.g.,the type 3 CPS of Streptococcus pneumoniae and EPS structuressuch as alginate, cellulose, and hyaluronan (40, 41). In Salmonellaenterica serovar Borreze O:54, a synthase is responsible for O-an-tigen biosynthesis (42). Synthases are relatively small integralmembrane proteins consisting of 4 transmembrane domains anda large cytoplasmic loop. This loop harbors the catalytic residuesand a Q-X-X-R-W motif involved in the processivity of the en-zyme by retaining the growing polymer in the enzyme (43, 44).The synthase of S. pneumoniae loosely associates with the growingglycan chain in the beginning but interacts more tightly after syn-thesis of a short oligosaccharide to ensure processive elongation(45).

Priming GTs are implicated in the en bloc biosynthesis of glyco-conjugates such as EPS, CPS, LPS O antigen, and glycoproteins.These are special GTs, as they do not catalyze the formation of aglycosidic bond. Priming GTs are linked to the membrane and areinvolved in the transfer of a phosphorylated sugar monomer froman activated nucleotide to an undecaprenyl phosphate (UndP)carrier, on which the glycan is then further assembled. These en-zymes, also known as initiating GTs, catalyze the formation of anenergy-rich phosphate bond (46–49). This UndP-linked sugarmonomer forms the substrate for the successive action of GTs thatbuild up the glycan further (see below).

OSTs transfer an oligosaccharide en bloc from the lipid carrierto the acceptor substrate, a protein (50, 51). The currently best-studied and best-characterized OST is the PglB OST of Campylo-bacter species (50, 52–55). This OST is strikingly similar to thecentral catalytic STT3 enzyme of the eukaryotic multicomponentOST counterpart (56). Bacterial OSTs, like PglB, however, aremonosubunit enzymes (57). PglB is involved as an N-OST in theN-glycosylation of proteins in Campylobacter species, and the fac-tors determining its catalytic mechanism and specificity have beenextensively studied. The catalysis of the oligosaccharide transferwas linked to the presence of an N-acetyl group in position 2 of thesugar directly linked to the UndP carrier. This C-2 acetamidogroup at the reducing-end sugar is necessary for recognition

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and/or catalysis either as a partner in forming critical hydrogenbonds with the OST or as a stabilizer of the transition state (53).The PglB enzyme was shown to recognize a D/E-Y-N-X-S/T(where Y and X cannot be P) motif located in flexible loops offolded proteins, which is an extension of the eukaryotic N-glyco-sylation motif (N-X-S/T) (52, 58, 59). More specifically, D-Q-N-A-T was identified as being the optimal consensus sequence for C.jejuni (52, 60).

In the biosynthesis of polysaccharides (EPS, CPS, and LPS Oantigen), the OST is replaced by a polymerase (61, 62). In the caseof polysaccharide biosynthesis, several oligosaccharide buildingblocks are linked together to form the glycan. This reaction iscatalyzed by a “Wzy” polymerase (see below) (61–65).

In most cases, the factors determining the specificity of GTsremain elusive, and it is difficult to predict the glycoconjugatebiosynthesis pathways in which they are involved based merely onsequence analysis. Thus, a crucial area for further research in bac-terial glycobiology is the search for factors or motifs determiningthe substrate specificity of GTs, both in the GTs and in the sub-strates. This would significantly improve the annotation of GTsand glycoconjugate biosynthesis clusters and would also be highlyrelevant for ongoing bacterial genome and metagenome projects.A complicating aspect, however, is that some GTs, and especiallyseveral OSTs, show promiscuity toward different substrates. PglBof C. jejuni, for instance, can transfer both O-antigen polysaccha-rides and N-glycosylated proteins (57). Another example is theO-OST of N. meningitidis, PglL, which can interfere with PG bio-synthesis and even act as a Leloir GT by transferring monosaccha-rides (66–68).

Sequential Glycosylation

Sequential glycosylation (Fig. 1) in bacteria is the most basic formof glycosylation: GTs transfer the sugar moieties directly to thesubstrate and build up the glycan. Sequential glycosylation hasbeen documented for both Gram-positive and -negative species(see below). Essentially, GTs use nucleotide-activated sugars asdonors to transfer sugar moieties to their substrate in the cyto-plasm. This can occur directly, as in the case of protein glycosyla-tion or glycosylation of TAs, or, in the case of CPS and LPS O-antigen biosynthesis, the glycan is synthesized while associatedwith the membrane (e.g., via diacylglycerol or UndP in rare cases).Once synthesized, the glycoconjugates are transported throughthe inner membrane (Gram-negative species) or cytoplasmicmembrane (Gram-positive species). Some peculiar GTs, i.e., syn-thases, are multifunctional and are responsible for both the bio-synthesis and the transport of the glycan (see above) (Fig. 1).

The glycans synthesized by this pathway are usually rather sim-ple, meaning that they consist of a single or only a few differentsugar building blocks. In many cases, several GTs work in concert,often even processively, i.e., without release of intermediate glycanproducts. Good illustrations of the sequential glycosylation mech-anism are the ABC transporter-dependent pathways of CPS (e.g.,Escherichia coli group 2 and 3 capsules [69]) and LPS O-antigen(e.g., E. coli 08 and 09a [63]) biosynthesis. Also, bacteria using asynthase for their CPS (e.g., type 3 CPS of S. pneumoniae [70]),EPS (e.g., alginate produced by Pseudomonas aeruginosa [71]), orLPS O-antigen (e.g., S. enterica serovar Borreze O:54 [42]) biosyn-thesis follow the sequential glycosylation mechanism describedhere. Substitution of proteins with simple glycans (both N- andO-linked), like flagellar glycosylation in several pathogenic spe-

cies, also illustrates the sequential mechanism neatly (72, 73). Fur-thermore, TA glycosylation in Firmicutes is thought to occur viathe direct attachment of sugars to the backbone (74). The wide-spread nature of sequential glycosylation in bacteria is illustratedin further detail below, where several type examples of bacterialglycoconjugate biosynthesis are discussed.

En Bloc Glycosylation of Glycoconjugates

The second important modular mechanism involved in thebuildup of glycoconjugates is the en bloc mechanism (Fig. 2). Thismechanism is essentially dedicated to the biosynthesis of morecomplex glycoconjugates. En bloc biosynthesis of glycans starts inthe cytoplasm, where a membrane-anchored priming GT utilizesa nucleotide-activated sugar residue to add a first glycan subunitvia an energy-rich pyrophosphate bond to a recyclable lipid car-rier (i.e., UndP) (46, 47, 75, 76). The glycan is then further assem-bled by the successive and concerted action of GTs that use nucle-otide-activated sugar residues as donors. Once a building block ofthe glycan is fully synthesized, the lipid-linked oligosaccharide isflipped or transported across the inner membrane (Gram-negativespecies) or the cytoplasmic membrane (Gram-positive bacteria) (46).Here, a polymerase links several building blocks and forms a repeti-tive glycan polymer, which leads to the production of EPS, CPS, andO-antigen LPS structures (63, 65). The flippase is commonly desig-nated Wzx, and the polymerase is designated Wzy (Wzy-dependent

FIG 1 The sequential mechanism of bacterial glycoconjugate biosynthesis. Inthis rather intuitive way of glycosylation, glycosyltransferases (GTs) transferthe sugar moieties from nucleotide-activated sugar donors directly to theirsubstrate. This substrate can be a glycoprotein or a growing polysaccharidechain linked to the membrane. Once the glycoconjugate is fully synthesized, atransporter transfers the structure to the periplasm (Gram-negative species) orto the other side of the cytoplasmic membrane (CM) (Gram-positive species).In some cases, the GT is also responsible for the transport of the glycan acrossthe membrane. Abbreviations: EPS, exopolysaccharide; CPS, capsular polysac-charide; LPS, lipopolysaccharide; WTA, wall teichoic acid; LTA, lipoteichoicacid; IM, inner membrane.



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pathway) (62, 64). In the case of glycoprotein biosynthesis, the poly-merase is “substituted” by an OST (see above), which transfers andligates the glycan to a protein substrate in an en bloc manner (50, 59).So far, this mechanism has been reported only for the O- and N-linked glycosylation of Gram-negative species, with N-protein glyco-sylation in C. jejuni being a type example (77, 78) (Fig. 2). The enbloc mechanism and detailed illustrations of bacteria using thispathway to synthesize their glycoconjugates are discussed furtherbelow.

BACTERIAL GLYCOCONJUGATES

Bacterial glycoconjugates display an enormous structural diver-sity. Bacteria can produce a variety of both intracellular and extra-cellular polysaccharides (Fig. 3.). The major glycoconjugates ofthe glycome of Gram-negative bacteria encompass PG, EPS, CPS,LPS, LOS, glycoproteins (both O- and N-linked), and intracellularstorage polysaccharides. Gram-positive organisms lack LPS andLOS but can have lipoglycans or glycosylated TAs.

Intracellular polysaccharides are used as an energy reserve, likethe well-known storage polysaccharide glycogen. An overview ofthe knowledge on glycogen and other bacterial intracellular stor-age polysaccharides is beyond the scope of this review, and thereader is referred to existing reviews (79, 80).

Peptidoglycan

In almost all Gram-positive and -negative bacteria, PG forms thebasis of the cell wall. PG is an enormous macromolecular het-eropolymer of �(1-4)-linked N-acetylglucosamine (GlcNAc) andN-acetylmuramic acid (MurNAc). The carboxyl group of the lat-ter is substituted with a peptide subunit, which is species specific.Neighboring glycans are linked via peptide cross-links to form astrong and rigid 3D structure. These peptide chains typically con-tain some D-amino acids, resulting in heightened resistance topeptidases (81, 82). This results in a mesh-like sacculus conferringcell support, shape, and strength to resist internal turgor pressure(82). The murein sacculus can be viewed as a bacterial exoskeleton(83). The maintenance of the integrity of the PG sacculus is essen-tial to cell viability, which is reflected by the numerous antibiotics

FIG 2 The en bloc mechanism of bacterial glycoconjugate biosynthesis. Inthe en bloc mechanism, in addition to standard Leloir glycosyltransferases(GTs), peculiar GTs come into play, i.e., the priming GT and oligosaccha-ryltransferase (OST) (glycoproteins) or polymerase (polysaccharides). Thepriming GT forms an energy-rich bond between a sugar-phosphate moietyand the undecaprenyl phosphate (UndP) carrier. Several GTs then consec-utively glycosylate the lipid carrier further, until the full glycan (in the caseof glycoproteins) or the building block (polysaccharides) is synthesized. Atransporter (e.g., a flippase) then transfers the glycans to the other side ofthe inner membrane (IM) (Gram-negative species) or cytoplasmic mem-brane (CM) (Gram-positive species). Glycosylated proteins are formedthrough the action of an OST, which transfers the glycan to the protein. Inthe case of polysaccharides such as exopolysaccharide (EPS), capsular poly-saccharide (CPS), and the O antigen of lipopolysaccharide (LPS), thebuilding blocks are assembled by a polymerase.

FIG 3 Bacterial glycoconjugates. Bacteria synthesize a plethora of glycoconjugates. Characteristic for Gram-negative species are lipopolysaccharide (LPS)and lipooligosaccharide (LOS) structures, while several Gram-positive species are known to glycosylate their teichoic acids (TAs) (wall teichoic acid[WTA] and lipoteichoic acid [LTA]). Both Gram-negative and -positive species can produce glycosylated flagella, pili or fimbriae, capsular polysaccharide(CPS), exopolysaccharide (EPS), and glycoproteins. The cell walls of both Gram-negative and -positive species also harbor a peptidoglycan (PG) layer,which serves as a scaffold for the anchorage of other molecules. The round dots represent proteins, and the triangles are ribitol phosphate or glycerolphosphate moieties.

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targeting PG biosynthesis. As the polymerization of PG takes placeoutside the cytoplasmic membrane, these processes are excellenttargets. Resistance to �-lactam antibiotics, acting at the formationof peptide cross-links, drives interest toward developing antibiot-ics targeting the transglycosylation step (35, 84). PG also plays arole in the recognition of bacteria by the host immune system(85).

The PG layer of Gram-positive bacteria is characteristicallythick, ranging between 15 and 30 nm (82, 86, 87), while in Gram-negative species, this layer generally measures only 7 to 8 nm, aswas experimentally shown for E. coli (82, 88). PG cannot be viewedas merely a rigid structure, as the macromolecule is subjected tocontinuous expansion and turnover (83). This turnover is carriedout by PG hydrolases, which, strikingly, are glycosylated in severalspecies (see below) (89–93). PG is also modified after assembly inmany species, and in Gram-positive species, the PG serves as ascaffold on which cell wall-associated molecules, such as TAs andCPS, are anchored (81, 83). Despite the numerous studies on PG,some questions still remain to be answered, such as the conforma-tion of the PG monomer subunits (94). A brief overview of PGstructure and biosynthesis is given, with a focus on the role oftransglycosylases, but for more detailed information, the reader isreferred to the reviews mentioned above.

PG biosynthesis is extremely conserved across species and waselucidated mainly in E. coli. Biosynthesis occurs following an in-side-outside growth strategy. Shape is maintained, as the biosyn-thesizing enzyme complex copies the preexisting murein sacculus(83). The general PG biosynthesis scheme involves two large steps(Fig. 4): the production of the lipid II PG precursor and thepolymerization of the precursors to form the PG layer. The for-mation of the lipid II PG precursor takes place in the cytoplasm byusing a series of UDP precursors and a UndP carrier. The succes-sive action of 6 enzymes, termed MurABCDEF, results in a UDP-MurNAc-pentapeptide precursor. The MraY enzyme catalyzes theformation of lipid I (phospho-MurNAc-pentapeptide coupled toUndP). MurG adds GlcNAc to lipid I, synthesizing lipid II, the PGprecursor. An unknown mechanism, potentially a flippase, trans-fers the lipid intermediate through the hydrophobic membrane tothe external side of the cytoplasmic membrane, where PG assem-bly takes place. In the second phase, lipid II is bound to the pre-existing cell wall material by the activity of transglycosylases andtranspeptidases (39) (Fig. 4). Polymerization proceeds essentiallyvia transglycosylation of the glycan strains and is followed by theformation of peptide cross-links. Both reactions are tightly cou-pled, which is illustrated by the bifunctionality of some penicillin-binding proteins (PBPs) harboring both transglycosylase andtranspeptidase activities. PBPs are a homologous family of highlyconserved membrane-bound proteins. The PG transglycosylases,both mono- and bifunctional ones, are all members of CAZy fam-ily 51 and show some homology to lysozyme. Transglycosylasesuse lipid II as an acceptor and the growing chain as a donor toform long PG glycan chains (30, 37). These enzymes work proces-sively, catalyzing multiple rounds of coupling without releasingthe elongating product. New lipid II units are successively addedto the reducing end of the growing polymer (36, 38). Biochemi-cally, transglycosylation is a nucleophilic substitution involvingthe displacement of undecaprenyl pyrophosphate (UndPP), re-sulting in configuration inversion (35, 39).

Organisms usually have multiple PBPs. This multiplicity re-flects the variety of functions that they have. Some PBPs are non-

essential and are involved in the rearrangement of PG in order toallow the assembly of envelope-spanning complexes and the per-meability of the cell wall (95). The number, molecular weight(MW), and abundance of PBPs present in a bacterium are speciesspecific. E. coli harbors 12 PBPs, with 5 having a high molecularweight and 7 having a low molecular weight. Low-molecular-weight PBPs are involved in cell separation, PG maturation, orrecycling (96).

The high-molecular-weight PBPs are crucial for PG biosynthe-sis and are subdivided into class A and B PBPs. Class A PBPs arebifunctional enzymes, harboring both transglycosylation (N-ter-minal) and transpeptidation (C-terminal) activities (95). Evolu-tionarily, these enzymes are believed to be the product of a fusionevent (97). These enzymes can act as a monomer or dimer viaN-terminal interactions (98). Class B PBPs have only one func-tional domain and catalyze transpeptidation events. Apart fromPBPs, membrane-bound, non-penicillin-binding, monofunc-tional GTs are also found, such as Mgt in E. coli. These GTs showsequence homology to class A PBPs and also use lipid II as a sub-strate. Monofunctional PG GTs are believed to catalyze the for-mation of un-cross-linked PG (35, 39).

Three high-molecular-weight PBPs of E. coli are PBP1a, PBP1b,and PBP1c. PBP1b is the best-studied one (97, 99) and, likePBP1a, is involved in the formation of glycan strands of 25 disac-charide units and peptide cross-links, starting from lipid II. Thesetwo PBPs are the major ones and have a semiredundant function(100). It is likely that the sugar units of both lipid II and the grow-

FIG 4 Peptidoglycan (PG) biosynthesis. PG is the scaffold on which manymolecules anchor. PG is itself a glycoconjugate, as it is a polymer of N-acetyl-muramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues. PGbiosynthesis utilizes an undecaprenyl phosphate (UndP) carrier, and the firststeps are carried out by the enzymes MurABCDEF, which add the firstMurNAc-phosphate residue and a pentapeptide onto the lipid carrier. TheMraY enzyme adds an extra phosphate, producing the substrate for the actionof the MurG glycosyltransferase (GT), which adds a GlcNAc, resulting in theformation of the lipid II precursor of PG biosynthesis. A transporter, poten-tially a flippase, then transfers lipid II across the inner membrane (IM) (Gram-negative species) or the cytoplasmic membrane (CM) (Gram-positivespecies). Penicillin-binding proteins (PBPs), which are (bifunctional) trans-peptidases and transglycosylases, finally incorporate the precursors into thePG layer (39). PG hydrolases are involved in the breakdown of the PG layer andare thus essential for PG turnover. Strikingly, these enzymes can be glycosy-lated, such as MspI of L. rhamnosus GG (89).



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ing PG chain are recognized by multiple sites of the PBPs (39). Therole of PBP1c is less well known, but this enzyme is also involved inmurein polymerization (101).

Rod-shaped bacteria harbor two PG-synthesizing complexes,one involved in elongation and the other one linked to septation.PBP1a and/or -1b is present in both complexes, while PBP2 andPBP3, monofunctional transpeptidases, are specific (39, 83).PBP2 is linked to elongation processes and is active in the presenceof PBP1b and PG (100, 102). PBP3 is located at the septum and islinked to cell division and septation (100).

In many species, the PG glycans are modified after insertioninto the cell wall. These modifications affect PG hydrolysis andenlargement during growth. Also, they increase resistance to hostdefense factors, like lysozyme, and decrease recognition by hostfactors. Common modifications are the N-deacetylation ofGlcNAc and MurNAc (which confers resistance to lysozyme), for-mation of �-lactam muramic acid, N-glycosylation of muramicacid in Actinomycetales, and O-acetylation of MurNAc in manypathogens. In Gram-positive species, TAs, lipoglycans, and CPSare linked to the glycan strands of PG via a species-specific linkageunit (81).

PG biosynthesis is closely intertwined with the cell division ma-chinery (100, 102). PBPs interact with many enzymes and struc-tural proteins, the most important ones of which are the divisomeand elongase complex of the rod-shaped cell. Together, all theseenzyme form a huge cell wall synthase complex (98, 100).

Cell Wall Polysaccharides: Capsular Polysaccharides andExopolysaccharides

Cell wall polysaccharides (CW-PSs) are large polymers of sugarmonomers, which are of great biological importance as they en-hance bacterial survival and modulate bacterium-host interac-tions.

Among bacterial CW-PSs, EPS and CPS can be distinguished.These structures differ in their degrees of attachment to the cellsurface. EPS structures are typically secreted in the extracellularmatrix or loosely associated with the cell surface via electrostaticinteractions. CPSs are linked more closely, often covalently, to thebacterial cell wall and can form a thick glycan layer, or capsule,around the cell. These CW-PSs can be further subdivided inhetero- and homopolysaccharides, according to their construc-tion, out of a mixture or a single type of sugar monomer. Thestructural variety of CPS and EPS molecules is enormous, evenbetween strains of the same species, and is based on the variationin sugar monomers, linkages, the presence of branches, and dec-oration with noncarbohydrate moieties. Also, the gene clustersresponsible for biosynthesis can be prone to phase variation (103).The unique character of CPS and EPS forms the basis of strainserotyping. For instance, in E. coli, more than 80 distinct capsularor K antigens can be discriminated, which are divided into fourgroups (62, 65, 104).

So far, three pathways for the biosynthesis of CPS and EPSstructures have been discriminated. The Wzy-dependent andABC transporter-dependent mechanisms are the most wide-spread (62). The third mechanism, a synthase-dependent system,has been only fragmentarily reported, e.g., for type 3 CPS assemblyin S. pneumoniae (70) and the biosynthesis of the EPS polyGlcNAcby, e.g., E. coli (105); alginate by P. aeruginosa (71); cellulose by,e.g., Salmonella (106); and hyaluronan by, e.g., Streptococcus pyo-genes (107). The O antigen of S. enterica serovar Borreze O:54 is

also produced via a synthase-dependent mechanism (42) (see be-low). CPS biosynthesis mechanisms are best studied for E. coli,where four groups of CPS structures are distinguished (see below).Under certain conditions (temperatures below 30°C), some E. colistrains also express an EPS, namely, colanic acid (65).

The Wzy-dependent mechanism is widely used by bacteria suchas E. coli group 1 and 4 members for CPS biosynthesis and bydiverse bacteria for EPS biosynthesis (64) (Fig. 5A). CPS en blocbiosynthesis starts in the cytoplasm, where single-repeat units aresynthesized. The first residue is transferred to a UndP carrier by apriming GT: WbaP, a polyisoprenyl-phosphate (polyisoprenyl-P)hexose-1-P transferase (49), or WecA, a polyprenyl-P N-acetyl-hexosamine-1-P transferase (48, 61, 108). Soluble or membrane-bound GTs complete the repeating-unit structure in a sequentialmanner. The repeating units are then flipped to the periplasmicside of the inner membrane or across the cytoplasmic membraneby a Wzx flippase (109). The Wzy polymerase builds up the CPS/EPS structure by the assembly of several repeating units at thereducing end of the growing polymer (61). The chain length of thegrowing CPS/EPS chain is determined by the reversible phosphor-ylation of the Wzc protein tyrosine kinase, which is capable oftransphosphorylation reactions (64, 110, 111). In Gram-negativespecies, a Wzb phosphatase is found in close proximity to the Wzcprotein, which can revoke phosphorylation again (111). The out-come of this reversible phosphorylation is still unknown, but itmight be related to the recycling of the UndP lipid carrier. InGram-positive species, a Wzb protein is often absent, but anotherphosphatase of the “polymerase and histidinol phosphatase” fam-ily takes over (e.g., CpsB in Streptococcus spp.) (112).

The second mechanism is called the Wzy-independent or ABCtransporter-dependent pathway (62, 69, 113) (Fig. 5B). Examplesare the CPS biosynthesis of E. coli group 2 and 3 members, N.meningitidis, C. jejuni, Haemophilus influenzae, and the polysialicacid (PSA) capsule of E. coli K1 (69, 114). The CPS chains are fullysynthesized in the cytoplasm prior to their transport across the cellenvelope. The early steps of sequential ABC transporter-depen-dent CPS biosynthesis are poorly understood: the initiation mech-anism remains unknown, and it is also not clear if a lipid carrier isinvolved in all species or not (62). Some organisms use Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) residues as a link betweenthe lipid and the sugar polymer (62). The KpsC and KpsS GTswere recently shown to catalyze this reaction (115). E. coli K1 CPSbiosynthesis involves a NeuS polysialyltransferase and a NeuE ac-cessory protein (116). The NeuD enzyme O-acetylates N-acetyl-neuraminic acid (Neu5Ac) prior to its incorporation into CPS(117). The initiating mechanism is unknown, but elongation oc-curs via the processive addition of Neu5Ac residues at the nonre-ducing end of the growing chain (118). The mechanisms govern-ing the chain length of these CPS structures remain to beidentified. Transport through the inner membrane requires anABC transporter composed of a homodimer of KpsM (the trans-membrane domain of the transporter) and a homodimer of KpsT(the nucleotide-binding part) (119, 120) (Fig. 5B).

Synthases, i.e., processive GTs, are enzymes central to the bio-synthesis of cellulose, hyaluronan, alginate, and polyGlcNAc aswell as the type 3 CPS of S. pneumoniae (40, 41). The type 3 CPS ofS. pneumoniae consists of an alternating chain of glucuronic acid(GlcA) and glucose (70) (Fig. 5C). The initiation, elongation, andtransport of the glycan are carried out by the Cps3S synthase andinvolve a phosphatidylglycerol lipid carrier. The first sugar mono-

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mer attached is glucose. Further polymerization at the nonreduc-ing terminus results in the final glycan structure. Two distinctkinetic phases can be distinguished in this process: during initia-tion, the enzyme associates loosely with the lipid-linked oligosac-charide until an eight-sugar-long oligosaccharide is produced. Forthe further processive elongation of this lipid-linked oligosaccha-ride, the substrate is bound more tightly, resulting in the fast andhighly processive synthesis of the polymer (41, 45) (Fig. 5C).Chain length is determined by the available concentrations ofUDP-GlcA, as UDP-Glc drives chain termination when inade-quate levels of UDP-GlcA are present (41, 121). In S. pyogenes, thehyaluronan CPS is formed via elongation reactions by the HasAsynthase. The initiation mechanism remains unknown but doesnot seem to require a lipid carrier (41). Alginate, a linear polymerof �(1-4)-linked mannuronic and glucuronic acids, is an EPS pro-duced by Pseudomonas aeruginosa and other Gram-negative spe-cies (71). The putative synthase is Alg8, and Alg44 forms a copo-lymerase, i.e., a receptor in the inner membrane that binds cyclicdi-GMP (c-di-GMP) and activates polysaccharide production(40) (Fig. 5D).

In Gram-negative species, the EPS/CPS structures are trans-ported after biosynthesis to the outer membrane by one or twodistinct outer membrane proteins. It is important to note thatthese mechanisms have to preserve the essential barrier propertiesof the envelope. The exact mechanisms of export are not entirelyclear, but the current knowledge has been summarized in a recentreview (122). In E. coli, K30 CPS structures are transported by Wza(Wzy-dependent mechanism), which is an acylated octameric�-helical protein barrel in the outer membrane that can adopt anopen or closed conformation. Wza is a member of the “outermembrane polysaccharide export” (OPX) family. The Wza trans-porter interacts with the Wzc protein in the inner membrane (Fig.5A). The Wzc protein is also required for polymer export inGram-positive species (62, 64, 123).

FIG 5 EPS and CPS biosynthesis pathways. (A) The en bloc Wzy-dependentpathway of EPS/CPS biosynthesis (illustrated for E. coli K30). EPS and CPSstructures can be synthesized in an en bloc manner, as in E. coli strain K30. Thisinvolves a priming glycosyltransferase (GT) (WbaP), which adds the first ga-lactose (Gal)-phosphate to the undecaprenyl phosphate (UndP) carrier. Thesuccessive action of several GTs (WbaZ, WcaO, and WcaN) results in theformation of the basic building block of the E. coli K30 CPS structure. A Wzxflippase transfers this building block to the periplasmic face of the inner mem-brane (IM), where a Wzy polymerase synthesizes the glycan polymer by con-necting the building blocks. The Wzc protein determines the chain length viaphosphorylation reactions, which can be reversed by the Wzb phosphatase.Wza, a member of the OPX (outer membrane export) family, further modu-

lates transport across the outer membrane (OM). The Wzi protein is a lectin inthe outer membrane, which interacts directly with CPS and facilitates capsuleformation (110, 123, 124, 166, 344). Abbreviations: Man, mannose; PG, pep-tidoglycan. (B) The sequential ABC transporter-dependent pathway of CPSbiosynthesis (e.g., E. coli K1). E. coli K1 capsules consist of a sequentially builtpolymer of N-acetylneuraminic acid (Neu5Ac). Their biosynthesis is probablyinitiated by the addition of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) res-idues by the KpsC and KpsS GTs. NeuS is the polysialyltransferase forming thepolysaccharide chain, while NeuE is an accessory protein. An ABC transporter,which is formed by a homodimer of the nucleotide-binding domain proteinKpsT and a homodimer of the transmembrane protein KpsM, facilitates ex-port through the inner membrane. KpsE is a membrane fusion protein, bridg-ing the inner and outer membranes. The KpsD protein is a member of the OPXfamily and ensures export of the glycan polymer to the outer side of the outermembrane (69, 113, 123, 344). (C) Sequential synthase-dependent pathway ofEPS/CPS biosynthesis (depicted for S. pneumoniae type 3 CPS). The CPS struc-ture of S. pneumoniae type 3 consists of alternating glucose (Glc) and glucu-ronic acid (GlcA) residues. Assembly is carried out on a phosphatidylglycerollipid carrier. Polymer initiation, elongation, and export are catalyzed by asingle enzyme, the Cps3S synthase (40, 41, 70). (D) Sequential synthase-de-pendent pathway of EPS/CPS biosynthesis (alginate biosynthesis). Alginate, apolymer of mannuronic acid (ManA) and GlcA residues, is biosynthesized bythe Alg8 residue, which needs the c-di-GMP-binding Alg44 copolymerase asan accessory protein. Several accessory proteins, such as AlgI, AlgJ, AlgF, AlgX,and AlgL, catalyze the O-acetylation of alginate. The AlgG epimerase catalyzesthe transformation of ManA residues to GlcA. The resulting glycan polymer isfurther transported by AlgK, which bridges the inner and outer membranes,and the AlgE outer membrane porin. The AlgL lyase degrades alginate in casethe AlgK protein is absent (40, 71).



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The K1 and K5 CPS capsules of E. coli, synthesized by the ABCtransporter-dependent pathway, require the KpsD protein for ex-port to the outer membrane (113). KpsD, an OPX family protein,like Wza, is not acylated and resides in the periplasm. Linkage tothe inner membrane is established via KpsE, a membrane fusionprotein, which allows the establishment of a bridge between theinner and outer membranes (62, 69, 123) (Fig. 5B).

The mechanisms of export across the outer membrane of syn-thase-dependent polymers remain largely unknown. Alginate isexported by a �-barrel porin in the outer membrane, AlgE. TheAlgK protein, a tetratricopeptide repeat (TPR) scaffold, forms abridge between the inner and outer membranes. Prior to export,the alginate is O-acetylated (AlgX, AlgI, AlgJ, and AlgF) in theperiplasm, and epimerization of mannuronic acid to glucuronicacid takes place (AlgG). AlgL, the alginate lyase, degrades alginatein the absence of AlgK (40) (Fig. 5D).

Surface association of CPS is established by the formation of acovalent bond between the glycan structure and a firm lipid an-chor and is carried out by the Wzi protein for E. coli group 1capsules. Wzi is a lectin, which interacts directly with the CPSstructure and assists in the formation of the capsule (62, 64, 124)(Fig. 5A). In the case of EPS, this association is loose and easilydisrupted. In Gram-positive species, the CPS is covalently an-chored to peptidoglycan by as-yet-unknown mechanisms (125).

Despite their immunogenic importance, many questions re-lated to their biosynthesis remain unanswered: the nature of theflippase and chain length determination system in Wzy-depen-dent systems; the initiation of polymer formation, the lipid an-chor, and the chain termination signal of ABC transporter-depen-dent systems; several aspects of synthase-dependent biosynthesisand export mechanisms; as well as details on the export and an-choring of CPS and EPS remain to be elucidated.

Functional roles of CPS and EPS encompass survival underharsh conditions (e.g., desiccation), the establishment of symbi-otic and pathogenic interactions with the host, protection of bac-teria from recognition by the host immune system, and biofilmformation (EPSs are important components of the extracellularmatrix) (126–130). Microbial CPSs are also of interest for thepharmaceutical industry, as the heterologous addition of CPSstructures to proteins can prolong the in vivo half-life of proteinssuch as insulin, which was modified via glycation (i.e., the nonen-zymatic addition of glycans) with polysialic acids (131). In thefood industry, lactic acid bacteria are used as starter cultures,mainly for their ability to produce EPS in situ. EPSs modulate thetexture and sensory qualities of food (132).

Bacterial CPS and EPS structures are protective structures andcan shield (immunogenic) surface molecules such as LPS inGram-negative species, lipoteichoic acid (LTA) in Gram-positivespecies, or invasion proteins. On the other hand, bacteria also useother surface molecules, such as adhesins, for interaction withtheir environment. The modulation of the balance between thesetwo functions is key for the successful interaction of bacteria withtheir host. However, not all EPS and CPS molecules form an im-munogenic shield. Depending on their exact composition andstructure, some CPS/EPS molecules are highly antigenic and canelicit strong antibody responses, resulting in the occurrence ofserotypes. Expression of CPS and EPS structures is thereforetightly regulated and prone to variation, contributing significantlyto EPS and CPS diversity (127). Also, in beneficial gut bacteriasuch as Lactobacillus rhamnosus GG, there seems to be an opti-

mum between EPS production for protection against innate im-mune factors and exposure of pili for optimal adhesion (133, 134).

In many bacteria, the expression of CPS genes can be turned onand off under the impulse of environmental stimuli, e.g., via ther-moregulation, such as the expression of colanic acid in some E. colistrains (65, 103). The biosynthesis of group 1 CPS and colanic acidin E. coli strains is regulated by the Rcs two-component regulationsystem, which lies upstream of the cps gene cluster. This systemconsists of RscC, a transmembrane sensor; RscB, the responseregulator; and RscA, the positive regulator of CPS expression.Homologues of this system are found in Klebsiella pneumoniaeand Salmonella species (65).

As indicated, CPS and EPS structures are often modified toavoid recognition by host immunity by altering the binding to,invasion of, and interaction with the host tissue. As mentionedabove, CPS and EPS structures, such as alginate, can show variousdegrees of O-acetylation (40). Phase variation is also common inCPS- and EPS-encoding operons (127). In C. jejuni, the CPS an-tigens are encoded in hypervariable regions of the genome (135,136). This genetic diversity of its CPS-encoding gene clusters re-sults in impaired host phagocytosis and complement-mediatedkilling (103, 130).

Antigenic diversity of the eight CPS regions of Bacteroides fra-gilis is modulated by mechanisms such as DNA inversion andtranscriptional antitermination (137, 138). Bacteroidetes spp. arealso known to incorporate glycans from the host into their surfacestructures, which enables an intimate and exclusive associationwith the host (139). This strategy of molecular mimicry is alsoused by species producing hyaluronic acid capsules and by N.meningitidis, which incorporates sialic acid into its CPS. In doingso, they inhibit complement activation and stimulate the pro-longed survival of the pathogen in the host (127).

From an immunological point of view, CPS and EPS structuresare quite peculiar. The fact that they are surface exposed makesthem ideal candidates for vaccine development, as this would in-duce the rapid phagocytosis of the pathogen. Problematically, CPSand EPS are antigens that elicit mostly T-lymphocyte-indepen-dent immune responses, with the exception of zwitterionic poly-saccharides (127, 140–142). Moreover, the wide variety of sero-types, which have variable disease-causing effects, and molecularmimicry of some polysaccharides make the development of vac-cines very challenging. Strategies to circumvent these limiting fac-tors are the coupling of the polysaccharide structure to a carriertargeting T-cell-dependent responses (T-cell memory) and theuse of peptides mimicking immunodominant structures (103,140, 142, 143). Hot topics in EPS/CPS vaccine development arevaccination strategies against N. meningitidis, H. influenzae type b,and S. pneumoniae (144).

Macroamphiphilic Glycolipids

Lipopolysaccharides and lipooligosaccharides in Gram-nega-tive bacteria. Seventy-five percent of the surface of the outermembrane of Gram-negative species is covered with LPSs or LOSs(145, 146). These macroamphiphiles are essentially built up out ofa lipid anchor on which long polysaccharide chains are anchored(147). LPS and LOS are essential for the survival of Gram-negativebacteria and are important immunomodulatory molecules (145,148). LPS molecules can be structurally subdivided into lipid A, acore oligosaccharide (inner and outer), and the O-antigen poly-saccharide. LOS molecules have largely the same structure but lack

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the O-antigen polysaccharide. LOS is found mainly in mucosalpathogens such as C. jejuni, Campylobacter coli, Neisseria spp., andHaemophilus influenzae (149).

The amount of research and papers on LPS and LOS structuresis enormous, which, in view of their abundance and importance, isno surprise. Here, we provide an overview of the biosynthesis ofthese structures, the O-antigen part in particular, and briefly dis-cuss their functional importance. For further reading, the reader isreferred to the vast numbers of research and review papers onthese topics, which are (partially) mentioned below.

Lipid A anchors the LPS structure in the outer membrane andcan elicit strong immune responses. It is the part that can provokeseptic shock as an endotoxin when it is shed from the outer mem-brane and interacts with Toll-like receptor 4 (TLR-4)/MD-2 com-plexes (150). The basis of lipid A in most bacteria is a 1,4-biphos-phorylated �(1-6)-linked D-glucosamine disaccharide substitutedwith covalently linked acyl chains. Lipid A can be further modifiedvia the addition of polar groups, phosphatidylethanolamine, andmodification of its fatty acyl chains (151, 152). Lipid A is highlyconserved, but minor differences in the amount, length, and link-age of the acyl chains and phosphates are possible. As mentionedabove, lipid A is very immunogenic, and modifications decreasethe endotoxicity of this structure (151, 153–158). For instance, ithas been shown that intestinal alkaline phosphatase reduces theproinflammatory potential of LPS of gut bacteria by removing thephosphate groups (159). In addition, many gut bacteria, such asBacteroides species, seem to have a less immunogenic lipid A struc-ture by having fewer acyl chains (160, 161). The hypoacylation oflipid A is also a strategy used by pathogens such as P. aeruginosaand N. meningitidis to decrease their immunogenicity (162, 163).

Lipid A is biosynthesized via the “Raetz” pathway, which takesplace in the cytoplasm and at the inner face of the inner mem-brane. Modification of the structure usually occurs outside thecytoplasm. In E. coli, nine conserved enzymes are involved in lipidA biosynthesis. LpxA is the first soluble enzyme of the pathwayand transfers a fatty acyl chain to GlcNAc. This UDP-monoacylGlcNAc is de-N-acetylated by LpxC in order to create a substratefor LpxD, which adds a second acyl group. The peripheral mem-brane protein LpxH (LpxI in many bacteria) cleaves the UMP atthe pyrophosphate bond and thus creates lipid X. This is con-densed with a UDP-2,3-diacylglucosamine to create the �(1-6)disaccharide. Integral membrane proteins such as LpxK producelipid IVA via phosphorylation, and in E. coli, this serves as a sub-strate for the addition of 2 Kdo residues by WaaL, which are part ofthe core saccharide. The lipid A structure is then further acylatedby the integral membrane proteins LpxL and LpxM (151).

The core oligosaccharide can be subdivided into an inner partand an outer part. The inner part is more conserved and consistsof Kdo residues and heptoses. In the outer part, hexoses are com-mon, and the structure is species specific. In E. coli, five core typesare found. Biosynthesis of the inner and outer core oligosaccha-rides occurs at the cytoplasmic face of the inner membrane. Thefirst residues of the inner core, 2 Kdos, are added during lipid Abiosynthesis and are required for the completion of lipid A. TheseKdos are further modified with heptose residues by the heptosyl-transferases WaaC and WaaF. WaaP phosphorylates the first hep-tose (HepI), and WaaX phosphorylates the second heptose(HepII). A third heptose is added by WaaQ (HepIII), which isfollowed by the addition of rhamnose (Rha) by WaaS and Kdo byWaaZ. The inner core can be further modified with phosphoetha-

nolamine and GlcNAc. The backbone of the outer core oligosac-charide of E. coli is synthesized by the GTs WaaG, WaaO, andWaaR and can be further substituted by the WaaT and WaaJ GTs.The addition of a glucose residue by WaaG is still a conservedreaction, while the reactions catalyzed by WaaO and WaaR, andespecially those catalyzed by WaaT and WaaJ, are more core typespecific (150, 164–166).

The lipid A-core is exported in E. coli by the MsbA membranetransporter and requires the presence of the 2 Kdo residues andcomplete acylation prior to export (150, 167). MsbA is the qualitycontrol step of LPS biosynthesis, as all transporters downstreamexhibit a more relaxed specificity (167).

The O antigen of LPS is the most diverse part of the LPS struc-ture: in E. coli, 170 different O polysaccharides (O PSs) have beenreported so far (168). This diversity is due to the composition,order, and linkage of the sugar residues in the repeating units and,together with the CPS structures (see above), determines theserotype specificity of strains (63). Also, phase variation in LPSO-antigen genes is common (156, 169). The O polysaccharide canbe neutral or contain negative charges, like phosphate and car-boxyl groups (170). These negatively charged residues help to sta-bilize the outer membrane by interaction with divalent cations(165). Repeating units typically consist of 2 to 6 residues and canbe linear or branched. Also, many unusual sugars are found in Opolysaccharides, of which the biosynthetic enzymes are encodedin the O-polysaccharide cluster (171).

O-polysaccharide biosynthesis, similarly to CPS biosynthesis,can follow three different mechanisms: (i) a Wzy-dependent path-way, (ii) an ABC transporter-dependent or Wzy-independentpathway, and (iii) a synthase-dependent pathway, which has, tothe best of our knowledge, been reported only in S. enterica sero-var Borreze O:54 (42, 63, 150). The first two pathways are wide-spread and are very similar to the biosynthesis of CPS and glyco-proteins.

The O polysaccharide is synthesized on a UndP carrier, whichis glycosylated with hexose-1-P (S. enterica) or hexosamine-1-P(E. coli) by a priming GT, WbaP or WecA, respectively (48, 49,108, 172, 173). The Wzy-dependent pathway is used in manyPseudomonas species and S. enterica for the biosynthesis of theO-polysaccharide heteropolymers at the inner surface of the innermembrane (Fig. 6A). In S. enterica serovar Typhimurium, the re-peating unit is a four-residue branched structure, synthesized onthe UndP carrier by the successive action of WbaP, WbaN, WbaU,and WbaV (150). After completion, the repeating unit is trans-ferred to the periplasmic side of the inner membrane by the Wzxtransporter. The Wzy enzyme then catalyzes the polymerization ofthe repeating units at the reducing end of the growing chain. Wzzis thought to be the chain length regulator, the mechanism ofwhich remains to be elucidated (63, 150) (Fig. 6A).

In organisms such as Bordetella species, some E. coli strains,Klebsiella pneumoniae, P. aeruginosa, Vibrio cholerae, and Yersiniaenterocolitica, the ABC transporter-dependent, or Wzy-indepen-dent, pathway is used for the biosynthesis of homo- and het-eropolymeric LPS O antigens. In E. coli serovars O8 and O9a, thispathway results in a homopolymeric mannose O antigen (Fig. 6B).After initiation by the priming GT WecA by the addition of Glc toa UndP carrier, the O antigen is further built up from mannoseresidues added to the nonreducing terminus of the growing oligo-saccharide by the WbdA, WbdB, and WbdC GTs. WbdD is theterminating enzyme and thus regulates chain length by methylat-



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ing the glycan: the higher the concentration of this enzyme, theshorter the glycan chains (174). An ABC transporter formed bythe Wzm-Wzt heterodimer transports the structure to theperiplasm (63, 150, 175–177) (Fig. 6B).

S. enterica serovar Borreze O:54 synthesizes its O antigen byusing a synthase-dependent pathway (42, 178, 179) (Fig. 6C). Itsantigen is a homopolymer of ManNAc residues with alternating�(1-3) and �(1-4) linkages. Initiation of glycan chain biosynthesisoccurs via the addition of a GlcNAc-1-P residue to a UndP carrierby the priming GT WecABorrezeO:54 (42). After the addition of thefirst ManNAc residue by WbbE, WbbF polymerizes and concom-

itantly exports the nascent O PS chain. WbbF is thus a synthasesimilar to the synthases involved in the biosynthesis of CPS struc-tures such as alginate (42) (Fig. 6C).

Both lipid A-core oligosaccharide and the O polysaccharide aresynthesized in the cytoplasm. After transport to the periplasm, theWaaL enzyme catalyzes the ligation of both structures via a cova-lent bond to form the LPS structure on the periplasmic side of theinner membrane (Fig. 6D). WaaL specifically recognizes the coreoligosaccharide but has a relaxed specificity toward a variety of OPSs. The O PS can be further modified by acetylation and glyco-sylation, and the anomeric linkage of repeating units can also be

FIG 6 O-antigen LPS biosynthesis and LPS assembly and transport. (A) O-antigen LPS biosynthesis via the Wzy-dependent en bloc mechanism (illustrated forSalmonella enterica serovar Typhimurium). Similar to the Wzy-dependent mechanism used for the biosynthesis of EPS and CPS in some structures, severalbacteria also use this pathway to form the O antigen of their LPS molecules. In S. enterica serovar Typhimurium, the priming glycosyltransferase (GT) is WbaP,which transfers a galactose (Gal)-phosphate to an undecaprenyl phosphate (UndP) carrier. The successive action of the WbaN, WbaU, and WbaV GTs results inan O-antigen building block. This is flipped across the inner membrane (IM) by the Wzx flippase and polymerized by the Wzy polymerase. The Wzz proteincontrols the chain length of the LPS O antigen (150, 179). Abbreviations: Rha, rhamnose; Abe, abequose. (B) O-antigen LPS biosynthesis using the sequentialABC transporter-dependent pathway (depicted for E. coli O8 and O9a). The E. coli O8 and O9a O-antigen LPS consists of a chain of mannose (Man) residuesattached to an initiating N-acetylglucosamine (GlcNAc) residue. The latter is added to a UndP carrier by the action of the priming WecA GT. The action of theWbdA, WbdB, and WbdC enzymes results in a mannose glycan polymer, which is terminally methylated by WbdD. Export to the periplasm is catalyzed by ahomodimer of both Wzt and Wzm, forming an ABC transporter (150, 179). Me, methyl group. (C) The rare synthase-dependent sequential O-antigen LPSbiosynthesis pathway of Salmonella enterica serovar Borreze. The N-acetylmannosamine (ManNAc) glycan polymer constituting the O antigen of the LPS of S.enterica serovar Borreze is initiated by the addition of a GlcNAc by the priming WecA GT. WbbE catalyzes the addition of the first ManNAc residue. The glycanchain is further extended and exported by the synthase WbbF (42, 178, 179). (D) Schematic representation of LPS assembly and transport. After assembly of thebasic parts of the LPS molecule (i.e., the lipid A-core oligosaccharide structure and the O antigen), these structures are ligated by the WaaL ligase. The LPSmolecules are then transported to the cell surface (i.e., the outer face of the outer membrane [OM]) by the Lpt proteins. LptB is an ABC transporter, and LptF andLptG are thought to provide energy for the transfer. LptC is the acceptor of the LPS molecule and transfers it to the LptA chaperone. The chaperone shuttles theLPS molecule to LptE, and finally, the LptD protein facilitates the transfer of LPS to the cell surface (150, 167, 179).

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altered (63, 150). Remarkably, LPS ligases and O-OSTs involvedin protein O-glycosylation (see below) are both members of thesame Wzy_C Pfam family (51).

Export of the complete LPS structure to the outer membranetakes place via the Lpt machinery, which spans the whole cellenvelope (Fig. 6D). The LptB, LptC, LptF, and LptG proteins arepresent in the inner membrane. LptB is an ABC transporter, whileLptF and LptG are believed to provide energy for the transporta-tion process via ATP hydrolysis. After transportation, the LPSmolecule is transferred from LptC (docking of LptA) to LptA,which acts as a chaperone, binds lipid A, and shuttles it to the outermembrane. LptD and LptE are enzymes in the outer membranethat are involved in the insertion of the LPS at its final destina-tion, but the exact mechanism remains to be elucidated (150,167) (Fig. 6D).

Functions of LPS include increasing cell wall integrity and pro-moting resistance against environmental stress, antibiotics, andkilling by the complement system and other innate immune fac-tors (150, 180). On the other hand, the LPS O antigen, togetherwith EPS/CPS, is one of the major antigens on the surface of E. coli,promoting adaptive immune responses by the host (170). There-fore, pathogenic strains often express a smooth version of theirLPS, which has a shorter or no O PS, or replace their LPS by LOSstructures to evade the strong host response provoked by the Oantigen (149, 154, 169). This is an irreversible transition and isestablished gradually over several generations (169). As men-tioned above, O PS can harbor unusual sugars, which in somecases mimic mammalian sugar structures. This form of molecularmimicry aids in the prolonged persistence of pathogens, as thebacteria can stay “stealth” from the immune system (181). An-other mechanism that assists in the prolonged presence of patho-gens is the phase variability of O-antigen genes, which results inantigen variation (156, 169). O-antigenic structures can also play arole in adhesion, for instance, in Salmonella Typhimurium, whereLPS is implicated in the adhesion and invasion of epithelial cells(182).

As mentioned above, lipid A is the part of LPS eliciting endo-toxicity (183). Lipid A interacts with almost all host cells but hasbeen investigated mainly in relation to monocytes and macro-phages (184). LPS-binding protein (LBP), a soluble acute-phaseprotein, binds LPS and shuttles it to the membrane-bound CD-14receptor (185). The CD-14 receptor facilitates transfer to the TLR-4/MD-2 receptor complex. Binding with this complex can inducethe MyD88-dependent (interaction at the cell surface) and -inde-pendent (interaction at the endosome) signaling pathways, bothof which can activate NF-�B. The result is the production of pro-inflammatory cytokines and type I interferons (148, 155, 158, 184,186, 187). LPS-neutralizing antibodies can inhibit immune re-sponses such as the TLR-4 dependent response elicited by lipid A,but the development of such molecules is hampered by a plethoraof serotypes. The inner core region is an interesting target fortherapeutic antibodies, as this region is more conserved (188–190). Recent antimicrobial approaches have focused on lipid Aantagonists, such as Eritoran, that can block these proinflamma-tory responses (191).

As stated above, many mucosal pathogens, such as C. jejuni andN. meningitidis, produce LOS, which shares the lipid A basic struc-ture with LPS but has an altered core oligosaccharide and lacks theO-antigenic polysaccharide (150). The altered core regions are lessconserved than LPS and are made up of short, nonrepeating oli-

gosaccharides. Like LPS, LOS structures are used for serotyping(149). Their biosynthesis has been best elucidated in C. jejuni 81-176 (192–196). Enzymes involved in the biosynthesis of LOS areprone to phase variation, which results in an enormous diversityof components and linkages (149, 192, 197–199). In C. jejuni, fivemechanisms of variation are common: variable gene comple-ments, phase variation via homopolymeric tracts, gene inactiva-tion by the deletion or insertion of 1 base, a single inactivatingmutation in a GT, or mutations leading to allelic GTs with analtered acceptor specificity (200).

LOS structures, like LPS, are often similar to antigenic struc-tures on human cells to evade host immunity (i.e., molecularmimicry) (149, 181, 192, 200). LOS structures can also be impli-cated in adhesion to the host (149, 201).

Lipoglycans in Actinomycetes. Gram-positive species with ahigh G�C content express macroamphiphiles on their cell wall,which can be compared to the LPS structures of Gram-negativespecies and TAs of low-G�C Gram-positive species (see below)(202). These macroamphiphiles are called lipoglycans and arefound in Actinomycetes such as Mycobacterium tuberculosis, Myco-bacterium smegmatis, Corynebacterium glutamicum, and Bifido-bacterium bifidum.

Corynebacteria have an extremely lipid-rich cell wall, as it con-sists of a complex of PG and arabinogalactan and is covered withnoncovalently linked lipoglycans. Three different lipoglycanstructures are found: phosphatidylinositol mannosides (PIMs),lipomannans (LMs), and lipoarabinomannans (LAMs). PIM is aprecursor for the biosynthesis of LM, and LM in turn is a precur-sor of LAM. PIM is in essence a phosphatidylinositol with a man-nose coupled to the inositol group. The PIM structure can bedifferentially acylated by acyltransferases, with up to four lipidgroups (203). To create LM, PIM2 (i.e., dimannosylated PIM) isfurther mannosylated with a glycan chain of 21 to 34 �(1-6)-linked mannose residues, which can be decorated with singular�(1-2)-bound mannose residues (204). If this structure is furthermodified with an arabinan domain, consisting of a linear chain of55 to 70 arabinose residues with branches at the reducing end, thestructure is called LAM (205–208). The long glycan chains of LAMcarry a terminal cap with either a monomer, dimer, or trimer ofmannose residues or a phosphatidylinositol (209, 210). Lipogly-cans from a single source are extremely heterogeneous in size,branching, acylation, modification with succinyl esters, and phos-phorylation (205, 209).

The biosynthesis of lipoglycans follows a sequential mecha-nism, as becomes clear from the fact that LAM structures are syn-thesized based on LMs, which are an extension of PIM structures.Several mannopyranosyltransferases are involved in the glycosy-lation of phosphatidylinositol (PimA, PimB=, PimC, and PimE),which can be further acylated by acyltransferases on the glycerol,myoinositol, or mannose moieties. Both PIM2 and PIM4 struc-tures can be further extended with linear mannan chains and arean acceptor of the MptB �-mannopyranosyltransferase. Furtherpolymerization in order to form LM results from the action ofMptA, MptD, and MptC (211). To create LAM, several arabino-furanosyltransferases (such as AftC, AftB, AftA, EmbB, andEmbA) are required (208). Mannopyranosyl capping of LAM inM. tuberculosis is carried out by a bifunctional mannosyltrans-ferase (Rv2181), which can both add �(1-2) branches to the back-bone and form mannose caps (212).

Important immunomodulatory roles are linked to lipoglycans:



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they can abrogate T-cell activity, inhibit gamma interferon (IFN-�)-induced functions such as macrophage activation, scavengefree radicals, inhibit protein kinase C, and evoke cytokines associ-ated with macrophages (205, 207, 209). Lipoglycans can bind tothe macrophage scavenger receptor (213), C-type lectins such asthe macrophage mannose receptor (207), and DC-SIGN, a C-typelectin receptor present on macrophages and dendritic cells (DCs)(214). Like other macroamphiphiles based on a glycosylated lipidanchor, such as LTA and some LPS molecules (e.g., Bacteroides),lipoglycans can be recognized by TLR-2 and stimulate the produc-tion of proinflammatory cytokines (148, 215). TLR-2 interactionis influenced by the acylation state of the lipoglycan and requiresthe interaction of TLR-2 with a coreceptor such as TLR-1 orTLR-6 (148).

Macroamphiphiles are also found in Bifidobacterium bifidum,Bifidobacterium breve, and Bifidobacterium longum strains. Theselipoglycans consist of a diacylglycerol anchor to which a glycanchain is linked. The glycan chain is bound to diacylglycerol via a�(1-3)-linked galactose, which is further polymerized by 8 to 12�(1-6) glucoses and 11 to 18 �(1-6)-linked galactose residues byas-yet-unidentified GTs. The terminal galactose residues are mod-ified with monomeric side chains of glycerophosphates, which arelinked via phosphodiester bonds. These glycerophosphates aresubstituted with L-alanine residues. This basic structure is com-mon to all bifidobacteria, but the numbers of polymers and re-peating units vary among strains (216).

Glycosylated teichoic acids in Firmicutes. TAs are anionicpolymers of poly(glycerol phosphate) or poly(ribitol phosphate)that are abundantly present on the cell wall of Gram-positive spe-cies with low-G�C-content genome sequences. Two types arediscriminated: wall teichoic acids (WTAs) and lipoteichoic acids(LTAs). The former are covalently linked to PG, and the latter areanchored to the cytoplasmic membrane via a lipid anchor. BothLTA and WTA can be modified with glycosyl and/or D-alanyl es-ters (217). The biosynthesis of TAs and their D-alanylation havebeen extensively studied and elucidated, so for more informationon these subjects, the reader is directed to specific reviews on thesesubjects (217–221), as they are beyond the scope of this work. Themechanisms and role of TA glycosylation still remain quite enig-matic and have been studied in only a few strains.

WTA usually extends beyond the thick PG layer, while it iscurrently believed that LTA remains in close proximity to the bac-terial membrane (217, 221). LTA is thought to reside in the Gram-positive bacterial periplasm (between the cell membrane and thePG), as was shown for B. subtilis 168 with nanogold-labeled anti-bodies (222).

Both WTA and LTA are important for bacterial physiology,and a considerable amount of energy is needed for the biosynthe-sis of these compounds. Both macroamphiphilic structures arepartially functionally redundant, which is illustrated by their sim-ilar tailoring modifications (D-alanylation and glycosylation),which also modulate their functions (220).

Both WTA and LTA are negatively charged, bind diversemono- and divalent cations to alleviate the electrostatic repulsionthat their negative charges create, and are therefore involved incation homeostasis (223, 224). Mutations of WTA and LTA alsoresulted in reduced biofilm formation, reduced adhesion to(a)biotic surfaces, and reduced virulence in many mucosal patho-gens, such as Enterococcus faecalis and S. aureus (221, 225–227).Analyses also point toward a role of the TAs in cell division and

elongation of Bacillus subtilis 168 (221). WTA plays a role in cellelongation in certain rod-shaped bacteria, as was shown by muta-tion of WTA, which resulted in a round bacterial morphologyinstead of a rod in B. subtilis (228). LTA is synthesized at the site ofcell division in B. subtilis and is believed to be involved in celldivision (229, 230).

Both LTA and WTA are implicated in bacterium-host interac-tions (227, 231). Their role in the activation of the innate immunesystem is still controversial, as contamination of the LTA sampleswith lipopeptides often cannot be ruled out. Nevertheless, highlypurified LTA molecules from, e.g., S. pneumoniae (232), L. rham-nosus GG (233), and S. aureus (234) have been shown to stimulatethe production of proinflammatory cytokines and NO and inter-act with TLR-2, while several coreceptors for the establishment ofthis interaction have also been reported: TLR-6, CD36, and CD14(148, 202, 231, 234, 235). WTA does not seem to be immunore-active like LTA, as has been shown, for example, in Lactobacillusplantarum WCFS1, where WTA appears to play mainly a shieldingrole (236). TAs are also believed to play a role in the evasion of theacquired immune system by shielding bacterial surface antigensand modifying the surface structure, which prevents antibodiesfrom binding (219).

The mechanisms and role of TA glycosylation in some bacterialspecies are less well known. Most advances have been made bystudying B. subtilis and S. aureus strains, and findings often datefrom several decades ago (Fig. 7.). Recently, renewed interest, al-beit still fragmentary, in the modification of TA has been ob-served, in view of the functional importance of (glycosylated) TAs(for example, in the search for new antimicrobials [237]) andtechnical advances in analyses.

FIG 7 Sequential glycosylation of TAs (WTA biosynthesis in B. subtilis 168).The biosynthesis of the WTA of B. subtilis 168 is initiated by the priming TagOglycosyltransferase (GT), which transfers an N-acetylglucosamine (GlcNAc)residue to an undecaprenyl phosphate (UndP) carrier. The TagA GT modifiesthis base further via the addition of an N-acetylmannosamine (ManNAc) res-idue. The TagB and TagF enzymes then work in concert to attach approxi-mately 40 glycerol-3-P units, which are glycosylated by the addition of Glc bythe TagE GT. TagGH is an ABC transporter transferring the glycosylated WTAto the outer side of the cytoplasmic membrane (CM) (74, 239, 240).

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Research on B. subtilis 168 established that the glucosylation ofits poly(glycerophosphate) WTA essentially requires the presenceof the following three enzymes: phosphoglucomutase (which pro-duces glucose-6-P from glucose-1-P) (238), UDP-glucose pyro-phosphorylase (formation of UDP-glucose) (238), and a UDP-poly(glycerol phosphate) �-glycosyltransferase, TagE (GtaA orRodD), which transfers the UDP-glucose to the C-2 position ofglycerol in the TA substrate. TagE is thus the GT targeting WTAand modifies the structure with single glucose monomers in aprocessive manner (74, 239) (Fig. 7).

The substitution of WTA with glucose residues probably oc-curs prior to transport (TagGH ABC transporter) out of the cell(74) and is thought to act as a regulator of the length of WTA bypreventing the binding of the polymerization enzyme TagF (240)(Fig. 7). The glycosylation of WTA was found to be dispensable forthe growth of B. subtilis 168 (241) but plays a crucial role in theadsorption of phages to B. subtilis 168 (241), as tagE mutants dis-played phage resistance (242, 243).

Apart from the model B. subtilis strain 168 for WTA glycosyla-tion (Fig. 7), glycosylated TAs also occur in other B. subtilis strains.Iwasaki and coworkers suggested that the glycosylation of LTA inBacillus strains can even be used as a taxonomic trait. B. subtilisand B. licheniformis strains display LTA modified with �-GlcNAcand D-alanine residues, while B. coagulans and B. megateriumstrains produce LTA substituted with �-galactose residues. Also,in B. cereus, �-GlcNAc-modified LTA can be found, which origi-nates from a �-GlcNAc-UndP donor (244). WTA is also deco-rated with glycans and D-alanine residues in S. aureus strains. Thepathways and enzymes involved in the glycosylation of WTA arein this case also largely unknown. The GT involved in the transferof GlcNAc to WTA was identified and is encoded by the tarMgene. Antibody studies showed the occurrence of both �- and�-GlcNAc residues on the poly(ribitol phosphate) backbone ofWTA in several S. aureus strains (245–247). In some strains, suchas the Copenhagen and NVH-6 strains, both �- and �-GlcNAc-decorated WTA molecules are found (245). Further research onthe Copenhagen strain showed that these differentially glycosy-lated TAs were synthesized by distinct enzymes (248). For severalstrains, the genes encoding GTs and the functional importance ofthe GlcNAc residues decorating WTA were elucidated by studyingspontaneous phage-resistant mutants (249, 250). The sugarmonomers on the poly(ribitol phosphate) seem to play a role inthe binding of bacteriophages and antibodies to S. aureus strains(245, 250).

In S. aureus strain RN4220, the activity of TarM, an �-GlcNActransferase, was extensively studied. Its activity was confirmed invitro via recombinant expression (251). The absence of signal pep-tides and the soluble nature of TarM suggest a cytoplasmic local-ization, which points toward glycosylation occurring prior to thetranslocation of WTA by the translocase TagGH. These findingsare indicative of a processive glycosylation mechanism. Whetherthe glycosylation of WTA is linked to the biosynthesis of the back-bone or targets the fully synthesized WTA structure remains to beelucidated. Strikingly, homologues of TarM were found in otherbacteria. The TagE enzyme of B. subtilis, for instance, is homolo-gous to TarM (251). Future research will have to reveal if thispoints toward a common glycosylation mechanism of WTA struc-tures in bacteria (251). Depletion of TarM resulted in a phage-resistant phenotype and indicated that the �-GlcNAc residues areinvolved in the adsorption of siphovirus (252). Recently, it was

shown that �-GlcNAc residues present on the ribitol phosphatesubunits of S. aureus RN4220 WTA are involved in the resistanceof methicillin-resistant S. aureus (MRSA) to the action of �-lac-tam (247). The TarS GT was identified as the enzyme responsiblefor this glycosylation and was recognized as a new target to tackleMRSA (247).

In Listeria monocytogenes, the glycosylation of the poly(ribitolphosphate) backbone of WTA is used as a taxonomic trait to dis-criminate between serogroups and serovars. Their cell walls con-tain no or only very little EPS/CPS. The main components of thelisterial cell wall are PG, LPS, and TAs (253). The WTAs of sero-vars 1/2, 3, and 7 are decorated with GlcNAc or Rha residues,while GlcNAc is incorporated into the backbone of the WTAs ofserovars 4, 5, and 6, and the GlcNAc is again decorated with Glcand Gal residues (254).

In L. monocytogenes serotype 4b strain M44, the poly(ribitolphosphate) backbone of WTA is covered with GlcNAc, which inturn is decorated with Gal and Glc residues. The genes responsiblefor these Gal and Glc modifications are gtcA and a gltA-gltB cas-sette. The GtcA enzyme was shown to be essential for substitutionwith Gal, and the absence of this enzyme also resulted in a reduc-tion in the number of Glc residues (255). The modification ofWTA GlcNAc by GtcA with Gal was shown to be essential for theadsorption of phages (256). Strains of the 1/2a serotype encodehomologues of GtcA (256). The gltA-gltB cassette is the regionmainly responsible for modification with Glc residues (257).

Information on TA glycosylation in other species is scarce. InStreptococcus sanguis, the LTA poly(glycerol phosphate) backbonehas been shown to be modified with D-alanine residues and gly-cans. The glycosylation of LTA can be used as a taxonomic markerfor S. sanguis, as its (1-6)-bound mono-, di-, tri-, and tetra-�-D-glucosyl structures are unique (258–260). The LTA of Enterococ-cus species is similarly decorated with sugar oligomers, but theseare linked to the backbone via a (1,2)-bound structure (261). Theglycosylation of TAs has also been reported for some lactic acidbacteria such as L. plantarum (Glc on WTA) (236, 262) and Lac-tococcus lactis subsp. cremoris strain SK110 (Gal on LTA) (263).However, this is not a general trait for lactobacilli, since, e.g., theLTA molecules of L. rhamnosus GG are not glycosylated (202,233).

Taken together, the glycosylation of TAs was already studieddecades ago, which yielded only a fragmentary view. The mecha-nism, structure, and role of TA glycosylation remain largely enig-matic, as the main focus has been on the elucidation of their role inphage adsorption. In view of the abundance of LTAs and WTAs inthe Gram-positive cell wall, new insights into their glycosylationwould shed light on their importance in bacterial physiology.

Protein Glycosylation

In 1999, based on an analysis of the Swiss-Prot database, Apweileret al. estimated that about half of the proteins in nature are glyco-sylated (264). Although their existence in prokaryotic species wasrefuted for a long time, glycoproteins were first discovered in Ar-chaea in 1976 (265), and the occurrence of protein glycosylation inBacteria has already been well documented since the first reportsof Clostridium bacteria in 1975 (266, 267). The fact that bacterialprotein glycosylation was discovered “quite late” can be linked tothe absence of glycoproteins in the most frequently used labora-tory strains of E. coli and Salmonella species (268). Glycosylatedbacterial proteins have been characterized mainly in pathogens



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and include surface layer proteins, pilins, flagellins, and enzymes.Recent studies also reported the occurrence of glycoproteins inbeneficial bacteria such as the gut symbiont Bacteroides fragilis(269, 270) and Lactobacillus species (89, 90, 271).

The glycosylation of proteins can occur via the linkage of gly-cans to the hydroxyl group of a serine or threonine residue and insome cases tyrosine residues, commonly referred to as O-glycosy-lation, or the glycan can be linked to an asparagine residue, whichresults in an N-glycosylated protein. Occasionally, such as for theglycocin F protein of L. plantarum KW30, glycosylation is linkedto a cysteine, resulting in S-glycosylation (272, 273). Protein gly-cosylation processes can also be distinguished based on the mech-anism used to transfer glycans to the protein substrate, i.e., in asequential or en bloc manner. Sequentially glycosylated substratesare directly targeted by GTs transferring sugar moieties (Fig. 1).Often, these systems are dedicated, meaning that the genes encod-ing the protein substrate and the GT, as well as the necessarytransporters, are clustered. If the glycan structure is fully synthe-sized on a lipid carrier (UndP carrier) prior to collective ligationon the substrate, this is called en bloc biosynthesis (Fig. 2).

Protein en bloc glycosylation in prokaryotes resembles the eu-karyotic N-glycosylation processes partially. In eukaryotes, pro-tein glycosylation occurs in the lumen of the endoplasmic reticu-lum (ER), while in prokaryotes, which do not have organelles, thesugar nucleotide precursors are synthesized in the cytoplasm. Bio-synthesis of the glycan then occurs in close proximity to the cellmembrane, as priming GTs are membrane-bound enzymes. Afterbiosynthesis, the glycan is flipped across the cell membrane. InGram-negative species, the transfer of the glycan to the substratetakes place in the periplasm. Whether Gram-positive species alsohave a periplasm is still debated, but several studies point towardthe presence of a small periplasm between the cell membrane andPG layer (86, 87, 274, 275). Both eukaryotes and prokaryotes uti-lize lipid carriers to assemble the glycans: dolichol pyrophosphatein eukaryotic species and UndP in bacteria (276). The en blocglycosylation of proteins is catalyzed by an OST, which is typicallya multimeric protein containing up to eight transmembrane sub-units in eukaryotes, while bacterial OSTs appear to be monomericproteins (57). In eukaryotes, en bloc N-glycosylation occurs priorto folding, when the substrates are still flexible (59). In contrast,data from C. jejuni suggest that en bloc glycosylation of proteinsoccurs on flexible, surface-exposed parts of folded proteins (59).In addition, eukaryotes perform quality control of the N-glyco-proteins, while the glycans are generally also further modified ortrimmed in the ER and Golgi apparatus. To the best of our knowl-edge, quality control and trimming of glycans in bacteria have not(yet) been reported.

Both eukaryotes and prokaryotes can perform en bloc N-glyco-sylation and sequential O-glycosylation of proteins. However, inprokaryotes, sequential N-glycosylation and en bloc O-glycosyla-tion have also been reported (277–280), highlighting that the gly-cosylation possibilities in prokaryotes appear to be greater. Cur-rent knowledge indicates that N-glycosylation of proteins occursalmost exclusively in Gram-negative species, namely, in epsilon-proteobacteria and a few deltaproteobacteria (279). There hasbeen only one report of a potentially N-glycosylated protein inGram-positive bacteria, in particular the platelet aggregation-as-sociated protein of S. sanguis (281). Taken together, O-glycosyla-tion is a widespread phenomenon in bacteria, while N-glycosyla-tion of proteins seems to be more restricted. Glycosylation of

proteins in bacteria has been shown to modulate the physico-chemical properties of their substrates. Glycan modifications ofproteins can protect them against proteolysis (e.g., AIDA-I of E.coli 2787 [282]), confer extra stability (e.g., C. jejuni proteins[283]), enhance activity (e.g., gingipains of Porphyromonas gingi-valis [284]), and change the surface properties (e.g., pili of P.aeruginosa 1244 [285]). In addition, glycoproteins play a role ininteractions between bacteria and their surroundings. Often, gly-coproteins are important virulence factors and antigens involvedin adhesion events (e.g., AIDA-I of E. coli 2787 [286]), immunemodulation (e.g., Apa glycoprotein of M. tuberculosis [287, 288]),and evasion (e.g., the pili of N. meningitidis [66, 289, 290]). Pro-tein glycosylation is often also heterogeneous and dynamic: oneprotein can carry more than one type of glycan, and glycans can beincomplete or modified. In Clostridium botulinum, this heteroge-neity can manifest itself even between flagella of different isolates(291), which points toward a role for glycoproteins as a strain-specific barcode.

Genome sequencing of the food-borne intestinal pathogenCampylobacter jejuni revealed an unexpected capacity for this or-ganism to produce a wide variety of glycoconjugates, includingPG, LOS, CPS, and N- and O-linked sugars (114, 135, 292). Gly-cobiologists started to focus their efforts on C. jejuni as a toolboxto unravel the mechanisms of protein glycosylation. Gradually, C.jejuni became a model organism for protein glycosylation, as it canboth N- and O-glycosylate proteins by en bloc and sequentialtransfer, respectively. Especially, the enzymes for protein N-gly-cosylation, clustered in the pgl locus, show a remarkably high levelof conservation (77, 293) and can even be functionally transferredinto Escherichia coli (50), which was one of the early milestones ofglycoengineering. In contrast, O-glycosylation genes are less con-served and phase variable (294, 295).

En bloc N-glycosylation of proteins. N-glycosylation of pro-teins has been described for only a few species, and its occurrencein bacteria is believed to be rare. En bloc N-glycosylation has so farmainly been studied in detail in Campylobacter species (77), whileH. influenzae and Actinobacillus pleuropneumoniae harbor asequential N-glycosylation mechanism (296, 297). Recently,genomic analysis revealed the presence of an en bloc N-glycosyla-tion mechanism in some Helicobacter species (298). N-glycosyla-tion in Chlamydia trachomatis has been suggested, butinformation on the underlying mechanisms is lacking (299, 300).For a long time, it was also believed that Borrelia burgdorferi, thecause of Lyme disease, produced four N-glycoproteins (FlaA,FlaB, OspA, and OspB) (301, 302). A later report by Sterba andcoworkers refuted these findings and attributed the positive gly-cosylation signals to tightly bound culture medium glycoproteins(303). This illustrates how important it is to document the occur-rence of glycoproteins in bacteria by at least complementarymethods.

C. jejuni, a gastrointestinal pathogen of humans and a com-mensal in avian colons, produces a wide variety of N-glycosylatedproteins. The first evidence of the existence of an N-protein gly-cosylation gene cluster came from the identification of LPS-likebiosynthetic enzymes in C. jejuni 81-176. The glycans produced bythe gene products of this locus, named the pgl locus (16 kb), seemto be immunodominant, as their absence has been shown to resultin reduced reactivity with antisera and a loss of immunogenicity(304). One of the first-characterized glycoproteins of C. jejuni isPEB3 (CJ0289c), an important antigen of C. jejuni (305) which is

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glycosylated with a heptasaccharide of 1,406 Da that reacts withthe lectin soybean agglutinin (SBA) (5, 306). An asparagine resi-due of PEB3 was shown to be modified with one monomer of thespecial sugar D-bacillosamine (Bac) (2,4-diacetamido-2,4,6-tride-oxyhexose), five D-GalNAc residues, and a D-Glc branch (5). Theen bloc nature of this process was confirmed by mutational analy-sis of the pgl locus, as interference at any stage of oligosaccharidesubunit assembly resulted in a total loss of glycoconjugate forma-tion (307). The only feature that seemed to be dispensable was theaddition of the glucose branch of the heptasaccharide (307). Grad-ually, more evidence emerged on the presence of other N-glyco-sylated proteins in C. jejuni strains (mostly in strains 81-176 andNCTC 11168), confirming the general nature of the N-glycosyla-tion process, i.e., targeting a range of proteins (5, 306, 308–313). Intotal, more than 60 glycoproteins have already been identified inC. jejuni (5, 114, 309, 311).

The conserved pgl locus comprises all enzymes necessary forthe N-glycosylation of proteins. Its role was confirmed by thefunctional transfer of the locus and the AcrA (CJ0367c) periplas-mic glycoprotein into E. coli, which resulted in the production ofglycosylated AcrA proteins (50). The pgl cluster was also exten-sively studied in vitro, which, complementary to mutant analysis,enabled the characterization of all enzymes individually (314).

PglF (CJ1120c) is a C-6 dehydratase, which, together with theUDP-4-keto-6-deoxy-GlcNAc C-4-aminotransferase PglE (CJ1121c)(315), is involved in the biosynthesis of the Bac moiety (316).PglD, an acetyltransferase modifying the UDP-4-aminosugar, iscoupled to the reactions catalyzed by PglF and PglE (317). Muta-tion of this enzyme resulted in monoacetylated Bac, which can stillbe transferred into mutant strains (309). A complementing en-zyme was also found in the C. jejuni genome (307). Later, PglDwas found to be a multifaceted acetyltransferase, as it can targetintermediates of both O- and N-glycosylation processes (318)(Fig. 8A).

The priming GT or Bac 1-phosphorytransferase PlgC, an inte-gral membrane protein, then adds Bac to the lipid carrier (UndP).Its reaction is coupled to the action of the consecutive GTs PglA,PglJ, PglH, and PglI (319). PglA adds an �(1-3)-linked GalNAc toBac, and this disaccharide serves as a substrate for the action ofPglH, PglI, and PglJ (Fig. 8.). The latter GTs add the other fourGalNAcs in an �(1-4) manner (the first GalNAc is added by PglJ,and the latter three are added by PglH) and add a branching�(1-3) Glc (PglI) to the structure (5, 136, 307, 314). PglH wasfound to have polymerase activity, as it can transfer three GalNAcresidues consecutively (314). This processive polymerase harborsonly a single active site, and product inhibition seems to limit thenumber of sequential GT reactions carried out by PglH to 3. Reg-ulation of this enzyme thus occurs via a “counting mechanism” ofproduct accumulation and inhibition (320).

The protein encoded by CJ1130c (wlaB) transfers the resultinglipid-linked heptasaccharide from the cytoplasm to the periplasmand was renamed PglK. PglK is an ABC transporter that uses thehydrolysis of ATP as an energy source to flip the glycans (321)(Fig. 8A). Strikingly, expression of the pgl locus in E. coli revealedthe presence of two distinct interchangeable mechanisms to flipthe lipid-linked oligosaccharides across the membrane: one usingPglK and the other using the Wzx flippase of the LOS pathway.Moreover, PglK even complemented a defect in Wzx, although thetwo enzymes showed very little similarity, which suggests a relaxedspecificity of these enzymes (321). In Helicobacter pylori, a homo-

logue of PglK, Wzk, was identified as a flippase involved in O-an-tigen biosynthesis (322).

An OST termed PglB (CJ1126c) finally transfers the oligosac-charide from the lipid carrier to the substrate protein (5, 306) (Fig.8A; see also above). Structurally, this enzyme contains 11 trans-membrane segments and two relatively large periplasmic regionsapart from the periplasmic C-terminal domain (323). PglB con-tains a strictly conserved W-W-D-Y-G motif, which is requiredfor its activity in vivo (50, 54). Several X-X-D domains were alsofound: two in the C terminus, one in the first periplasmic region,and three more in the periplasm. These domains are typical for GTactivity and are thought to be involved in catalysis (see above).

The PglB OST recognizes a motif in the acceptor substratesimilar to the eukaryotic N-X-S/T sequence (324). Work byKowarik and coworkers further extended the consensus sequenceof PglB to D/E-Y-N-X-S/T, with Y and X being any amino acidexcept proline (52). Those researchers also showed the require-ment for a negatively charged side chain at the �2 position of theglycoproteins. Chen and coworkers (60) took these findings a stepfurther and assessed variable amino acids at the Y and X positionsof the glycan sequon for their impact on glycosylation efficiency.The Y amino acid of the sequence is preferably a glutamine, aspar-agine, or large hydrophobic moiety, while for the one at the Xposition, there is a preference for negatively charged amino acidssuch as lysine, arginine, alanine, and serine. This resulted in thepostulation of D-Q-N-A-T as the optimal acceptor sequence (60).The lipid-linked oligosaccharides also need to be transferred to theperiplasm, where PglB is active (325, 326). In contrast to eukary-otic OSTs, PglB catalyzes the glycosylation of folded bacterial sub-strates with a high efficiency, but the glycosylation sites of bacterialproteins are present predominantly in the flexible, surface-ex-posed parts of the folded protein (59). The acceptor region ap-pears to partially adopt an unstable �-helix conformation andform random coils (327). These findings were confirmed experi-mentally via a crystal study of the PEB3 glycosylated adhesin(313). Three key glycosylated residues are well exposed at the sur-face, which makes them accessible to PglB even in the folded stateof the protein. In this way, PglB can target the glycosylation siteswithout the need for local protein structure rearrangements (313).

Research on the specificity of PglB, PglC, and PglJ for the poly-isoprenol diphosphate carriers on which the oligosaccharide isbuilt revealed the preference for cis double-bond geometry and�-unsaturation, while the precise length of the polyisoprene is lesscritical (328). Li et al. later confirmed this relatively relaxed spec-ificity for the lipid carriers (323). Moreover, PglB recognizes anacetamido group at the C-2 position of the sugar directly linked tothe UndPP carrier (53). Bac, GlcNAc, GalNAc, and FucNAc allhave a C-2 acetamido group, which can partially explain the pro-miscuity of PglB (the enzyme can even transfer monosaccharides)toward the sugars that it transfers (53, 323).

Studies of the functional importance of N-glycans present onthe proteins of C. jejuni are still rather scarce. These glycans seemto modulate the functions of several specific proteins, which arenot the same for all proteins. Impaired glycosylation of the VirB10glycoprotein, a type IV secretion system glycosylated at two sites,for instance, resulted in reduced natural transformation (329).Mutations affecting the pgl locus resulted in altered phenotypes,such as decreased adhesion and invasion efficiency in vitro andabolishment of mouse and chicken colonization in vivo (5, 315,330, 331). The glycans were also hypothesized to play a role in



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immune evasion by masking primary amino acid sequences,based on studies with human antisera (304). Moreover, the termi-nal GalNAc residues of the N-linked heptasaccharides were shownto interact with the human C-type macrophage galactose lectin(MGL) receptor present on human dendritic cells and distinctmacrophage subsets, thereby modulating immune responses(332). A mutation in the pgl locus also resulted in enhanced ex-pression of the proinflammatory cytokine interleukin-6 (IL-6),again implying a shielding role for the glycans (332).

In addition to a role in host interactions, recent work byAlemka et al. underlines the importance of N-glycosylation forbacterial fitness, as mutation of pglB resulted in reduced growth inmedium supplemented with chicken cecal contents (283). Thisresult could be linked to the presence of proteases in this medium,

as inhibition of serine and metalloproteases resulted in restoredviability and a partial rescue of bacterial growth. This finding addsfurther support for a role of N-glycans in protection against pro-teolysis of surface proteins.

A similar en bloc N-glycosylation system has been discovered inHelicobacter canadensis based on genome analysis (298). Furthergenome analysis showed the presence of PglB orthologues in H.canadensis, H. pullorum, and H. winghamensis (333). H. pullorumhas two orthologues of pglB, pglB1 and pglB2, which are unrelatedand not located in a large locus involved in protein glycosylation,although the colocalization of pglD, pglE, and pglF orthologues ina small separate cluster was reported. The PglB1 enzyme cantransfer the C. jejuni heptasaccharide in E. coli but results in themodification of only two sites of the CJ0114 protein, instead of the

FIG 8 Protein glycosylation. (A) En bloc N-protein glycosylation in the model organism C. jejuni. The type example of en bloc protein glycosylation is the generalN-protein glycosylation system of C. jejuni. Prior to the start of the glycosylation process, UDP-N-acetylglucosamine (GlcNAc) is modified to N-diacetylbacillosamine (diNAcBac) by the action of PglF, PglE, and PglD. The priming glycosyltransferase (GT) PglC transfers this sugar moiety to an undecaprenylphosphate (UndP) carrier, followed by the successive actions of the PglA, PglJ, and PglH GTs adding N-acetylgalactosamine (GalNAc) and glucose (Glc) residues.Once finished, the glycan moiety is transferred across the inner membrane (IM) by the PglK flippase. Finally, the PglB oligosaccharyltransferase (OST) thenN-glycosylates the protein (see references 77, 78, and 293 and the references mentioned in the text). OM, outer membrane. (B) The peculiar sequential N-proteinglycosylation system of H. influenzae. In H. influenzae, the glycan moieties are directly attached to serines and threonines in the protein by HMW1C. Transpor-tation across the inner membrane relies on the Sec pathway, followed by further export to the cell surface by HMW1B (73, 296, 335). (C) Sequential O-proteinglycosylation of pili, flagella, and other proteins (illustrated for Fap1 of S. parasanguinis). Glycosylation of the Fap1 fimbrial protein of S. parasanguinis remainsto be fully elucidated. It is currently known that the first step of the glycosylation process is catalyzed by a heterodimer of Gtf1 and Gtf2, while Gtf3 regulates thelatter glycosylation steps. The glycoprotein secretion mechanism is especially of note here, as it involves a SecA2-SecY2 heterodimer. SecY2 forms a translocationchannel, with SecA2 being an accessory protein. Gap1 and Gap3 are crucial accessory proteins for fimbrial biogenesis (399, 400, 402, 403, 406, 407, 409, 411, 412).(D) The general en bloc protein glycosylation system of Neisseria spp. In Neisseria spp., the PglD, PglC, and PglB or PglB2 enzymes modify a nucleotide-activatedGlcNAc residue. The action of PglB results in the formation of 2,4-diacetamido-2,4,6-trideoxyhexose (DATDH), and the action of PglB2 results in the formationof 2-acetamido-4-glyceramido-2,4,6-trideoxyhexose (GATDH). These enzymes are also the priming GTs, adding these sugar residues to a UndP carrier. Thesuccessive action of PglA and PglE results in the addition of two Gal residues. The glycan is then transferred to the periplasm by a PglF flippase and ligated to theprotein by the PglL/PglO OST (66, 67, 476, 480, 496). Gal, galactose.

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four sites that are modified by PglB of C. jejuni. This indicates theOST character of this enzyme and the distinct specificity com-pared to that of PglB of C. jejuni. PglB2 seems to be essential, as noknockout of this gene can be made (333). The character, activity,and functionality of this glycosylation system remain to be furtherinvestigated. At the moment, it seems that, in contrast to the find-ings in C. jejuni, the N-glycosylation of proteins is not a genus-wide feature of Helicobacter (333). Moreover, the only report of aglycoprotein in a Helicobacter sp. was on the glycosylation of RecAin H. pylori. Based on mutational analyses of recA and eno (anenolase homologue), glycosylation was claimed to be required forthe full functionality of RecA in response to DNA damage (334).However, glycosylation of RecA needs to be further substantiated,especially in other bacterial species, given its universal role.

Sequential N-glycosylation of proteins. For H. influenzae, adistinct mechanism of N-glycosylation was reported, in which gly-cosylation occurs in a sequential way (Fig. 8B). The high-molec-ular-weight HMW1 adhesin, which is loosely associated with thebacterial surface and involved in the attachment of the pathogento human epithelial cells, is decorated with galactose, glucose, andmannose residues (73). The glycan is believed to account for 5% ofthe total mass of HMW1 and is attached in the cytoplasm (73).Thirty-one sites of the protein are modified with mono- or di-hexoses (total of 47 hexoses), all harboring the N-X-S/T sequence,except for one, which is glycosylated at an N-V-E sequon (335).

HMW1 is associated with two accessory proteins, of whichHMW1B is involved in the translocation of HMW1 from theperiplasm to the cell surface and HMW1C was found to be re-quired for HMW1 glycosylation (73). The HMW1C proteinshows homology to GTs and can transfer glucose and galactoseresidues to HMW1. In addition, the GT can also generate hexose-hexose bonds, which suggests that this multifunctional GT be-longs to a new family of bacterial GTs (296, 335). HMW1C trans-fers glucose residues to all N-glycosylation sites, while it cantransfer galactose to only a subset of the glucosylated N-residues.How the HMW1C GT makes a distinction between both typesremains to be elucidated (296) (Fig. 8B). The biosynthesis of theUDP-glucose precursors that are linked to HMW1 requires anenzyme of the LOS pathway, namely, the phosphoglucomutasePgmB (73).

Glycosylation of the HMW1 adhesin was shown to play a rolein the tethering of the adhesin to the surface and stability of theprotein, as glycosylation defects result in an HMW1 adhesin that isprematurely degraded and released from the surface (73).

A homologue of the HMW1C GT of H. influenzae was found inActinobacillus pleuropneumoniae by the group of Aebi. This in-verting cytoplasmic N-glycosyltransferase, termed NGT, targetsthe N-X-S/T consensus sequence and modifies this sequon withGal and Glc residues. Elongation of the Glc residue can be per-formed by an �-1,6-glucosyltransferase (297). The relaxed speci-ficity of the NGT enzyme was illustrated by the reconstruction ofthis glycosylation pathway in E. coli. This general glycosylationsystem seems to have a preference for autotransporter adhesinsbut can also glycosylate heterologous proteins (336).

For Chlamydia trachomatis, which causes chlamydia in hu-mans, three N-glycosylated proteins were reported. The analysesall date from the early 1990s and relied on the use of lectins, gly-cosidases, and blocking experiments. The 18-kDa and 32-kDa lec-tin-binding proteins of the L2 serovar were found to be glycosy-lated, and the glycans were suggested to be of importance for the

interaction with HeLa cells (300, 337). The best-documented gly-coprotein is the major outer membrane protein (MOMP), whichconstitutes up to 60% of the chlamydial protein envelope. MOMPharbors the N-X-S/T sequence, and its N-glycans mediate the at-tachment and infectivity of C. trachomatis in HeLa cells (299, 338,339). This glycan is a high-mannose-type oligosaccharide of 8 to 9mannose residues. The presence of other sugar monomers, such asGlcNAc, was also suggested (339). However, these findings re-main to be further substantiated to ascertain that the results didnot originate from cross-contamination with human serum.

Dedicated O-glycosylation of proteins: flagella. C. jejuni hasbipolar flagella involved in the colonization of mucus (340, 341).The structural proteins FlaA and FlaB of C. jejuni flagella are dec-orated with glycans. In C. jejuni 81-176, up to 19 sites of a surface-exposed region of FlaA are glycosylated (342). This implies therequirement of surface-accessible S and T residues for the attach-ment of glycans (342). The glycan-modifying FlaA protein inmany C. jejuni strains, such as 81-176, is pseudaminic acid (Pse), a9-carbon sugar similar to sialic acid, or derivatives thereof (343).The flagellin structural protein FlaA of the related Campylobactercoli strain VC167 was found to be glycosylated on 16 sites, bothwith Pse and with legionaminic acid (Leg), which is a C-9 sugarrelated to sialic acid, like Pse, or its derivatives (344, 345). Whethera Campylobacter strain produces Pse, Leg, or both and which de-rivatives it produces are strain specific and determined by theheterogeneous O-glycosylation loci that it harbors. Flagellin gly-cosylation is a factor determining variety between strains and theirserospecificity (346, 347). FlaB is thought to be glycosylated sim-ilarly to FlaA in several C. jejuni strains (72).

The genetic locus responsible for the glycosylation of flagella inC. jejuni NCTC 11168 comprises nearly 50 genes and encompassesthe genes involved in Pse biosynthesis, Maf (motility accessoryfactor) genes, genes encoding the structural subunits FlaA andFlaB, and many genes with unknown function (135, 292). Of note,the related C. jejuni strain 81-176 has only half the number ofgenes, namely, 24 genes, involved in flagellar O-glycosylation(343).

Genes orthologous to sialic acid biosynthesis genes regulate Psebiosynthesis (neuA2, neuB3, neuB2, neuC2, and neuA3) (316,348). Leg biosynthesis is carried out by 11 ptm genes and usesparticular GDP-linked sugar residues (349). The maf genes (maf1to maf7) are members of the 1318 motility accessory factor familyof flagellin-associated proteins, and some (the identical maf1 andmaf4 genes) harbor polymorphic G tracts, which enable motilityvariation by slipped-strand mispairing (294, 295). The maf5 geneis involved in flagellum formation and is therefore invariant (294).Another four paralogous genes are members of the 617 gene fam-ily and contain intragenic single nucleotidic repeats, which areprone to phase variability. The exact role of the genes of both theCj1318-maf and 617 gene families (i.e., families of highly homol-ogous genes in the O-glycosylation cluster) remains to be eluci-dated (293). Strikingly, a GT activity has not yet been attributed toany of the 50 genes of the O-glycosylation cluster. So far, to thebest of our knowledge, there have been no reports on the charac-terization of one or more O-GTs in C. jejuni strains. Recently, ourgroup designed an in silico strategy to predict/annotate genes en-coding GTs in bacterial species more accurately (569). This studyidentified four candidate genes involved in O-protein glycosyla-tion in C. jejuni NCTC 11168, namely, Cj1328, Cj1329, Cj1331,



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and Cj1333. Further experimental validation of these targets isneeded to confirm that they are truly flagellar GTs.

The genetic variation displayed by both gene families (617 andCj1318-maf) results in an alteration in the behavior of C. jejunistrains. For example, phase variation of the maf4 gene of C. jejunistrain 108 results in altered glycosylation of the flagella and agglu-tination behavior (295). Other genes, such as Cj1295 of C. jejuniNCTC 11168, also contain single nucleotide repeats and ho-mopolymeric tracts, which can result in considerable structuralchanges in the flagellar glycoproteins (350).

In C. jejuni 81-176, glycosylation was shown to be required forflagellar filament assembly, as the glycans mediate interactionsbetween subunits within the filament (351). The flagellar glycansof strain 81-176 can also establish interactions between differentfilaments and modulate autoagglutination events (343, 351). Dif-ferences in glycan modifications on flagella impact the formationof microcolonies on intestinal epithelial cells in vitro, which iscritical for the virulence of strains (352). In C. jejuni NCTC 11168,flagellar glycosylation with Leg residues results in a change of thesurface charge, which is important for the colonization of the co-lon of chickens (353). Of note, the flagellar glycans of C. jejuniNCTC 11168 are not implicated in the evasion of recognition of C.jejuni flagella by TLR-5 (354). Mutation of genes involved in fla-gellar glycosylation results in a loss of motility in C. jejuni and C.coli strains (72, 355, 356).

H. pylori, a species closely related to C. jejuni, also producescomplex polar flagella consisting of two structural glycosylatedproteins, FlaA and FlaB. Both flagellins are modified with Pse:FlaA on 7 sites and FlaB on 10 sites (357, 358). The glycosylationsites lie in the central core region of the flagellin monomers, whichare surface exposed in the filament (358). In contrast to the re-markable heterogeneity of flagellin glycosylation in C. jejunistrains, the level of heterogeneity of isoforms and glycoforms islow in Helicobacter species. For instance, no derivatives of Pse havebeen reported. The sheath surrounding the flagellar filaments ofHelicobacter pylori probably negates the need for this variation(358). This results in a glycosylation region that is analogous tobut simpler than that of C. jejuni. This explains the large numberof functional studies of enzymes involved in Pse biosynthesis in H.pylori, which facilitated the elucidation of the C. jejuni mechanism(316, 359–361). The enzyme encoded by HP_0840 (PseB) wascharacterized as a bifunctional C-6-dehydratase/C-5-epimerase(316, 362). The HP_0366 gene codes for the flagellar aminotrans-ferase PseC (316, 360). Further elucidation of Pse biosynthesis inH. pylori revealed the function of the other enzymes involved:HP_0326A or PseF is the CMP-Pse synthetase; HP_0326B or PseGis the nucleotidase; PseH, encoded by HP_0327, is an N-acetyl-transferase; and the HP_0178 gene codes for the Pse synthase(PseI) (359). It was also shown in vitro that the modification ofUDP-GlcNAc to form CMP-linked Pse occurred in one step com-bining these six enzymes (359). The enzyme encoded by HP_0114is central to flagellar assembly, either via transfer of Pse to theflagellin monomer or via its function in later steps of flagellarassembly (358). The modification of FlaA with glycans at sevensites was proven to be a general feature of H. pylori strains (361).

Glycosylation of H. pylori flagellin is crucial for filament assem-bly and bacterial motility, as mutations in the glycosylation regionresulted in the absence of functional flagella (358, 363, 364).Flagellin is thought to be glycosylated only upon secretion (363).Study of a hypermotile mutant of H. pylori G27 resulted in the

identification of HP_0518 as an enzyme involved in the deglyco-sylation of FlaA (364). This mutant showed increased glycosyla-tion of FlaA, which points toward a link between its glycosylationstate and the pathogenicity of H. pylori, as this mutant can interactbetter with host cells and causes accelerated host cell responses,which is of course detrimental to a pathogen (364). This alsopoints out the need for strict regulation of glycosylation. Recentpapers reported the presence of other glycoproteins, many ofwhich are cytoplasmic, in H. pylori strains, besides the well-knownflagellar ones (365, 366). A mass spectrometry (MS) screening ofenriched azide-labeled proteins of H. pylori 26695 resulted in theidentification of 125 candidate O-linked glycoproteins having di-verse functions and located in different fractions of the cell (366).

In P. aeruginosa, two major serogroups (types a and b) arediscriminated based on the expression of the flagellin structuralprotein FliC. Totten and Lory reported an important discrepancyin molecular mass for FliC of P. aeruginosa type a strain PAK (52kDa instead of the calculated 45 kDa) (367). Eight years later, thismolecular mass shift was attributed to the glycosylation of type aflagellin (368). Genome comparison of type a strain PAK and typeb strain PAO1 revealed a flagellin glycosylation island of 14 genes(orfA to orfN) organized into several operons in strain PAK (369).In type b strain PAO1, this glycosylation island is replaced by threegenes of unknown function and a putative GT. This putative GTshows a low level of homology to the orfN gene present in PAK,which was recognized as a putative flagellin-GT (370). Furtherresearch on the glycosylation island in type a strains revealed ahigh level of polymorphism for this cluster. Many strains encode ashorter version of the island, in which orfD, orfE, and orfH arepolymorphic and orfI, orfJ, orfK, orfL, and orfM can be absent(371).

P. aeruginosa type a flagellins were shown to be modified at twosites (T189 and S260) on each monomer and produce uniqueglycan structures. The PAK flagellin is glycosylated with a hetero-geneous O-linked glycan, which can comprise up to 11 monosac-charides, linked to the protein backbone via a rhamnose residue.Flagellin of another strain, JJ692, has less complex modifications:single rhamnose subunits are linked to each site. In strain PAK, along glycosylation island is involved in the glycosylation of thePAK flagellin, encoding the OrfA enzyme responsible for the at-tachment of the heterogeneous glycan and the OrfN rhamnosyl-transferase. In contrast, strain JJ692 harbors a truncated glycosy-lation island with a length similar to those of the shorterglycosylation islands found in other Pseudomonas strains. Never-theless, these other strains can show more extensive glycosylationof flagellin than the single glycan substitution in JJ692. As in C.jejuni flagellar glycosylation, genetic variation of the glycosylationisland can result in altered glycosylation profiles (372).

It was believed for a long time that b-type flagella were non-glycosylated, until it was reported that the short genomic island (4genes) of PAO1 modifies S191 and S195 with a simple glycan of700 Da. This glycan was identified as a deoxyhexose with a uniquemodification of 209 Da, which harbors a phosphate moiety. Incontrast to a-type flagellar glycosylation, the level of polymor-phism of the glycosylation island is low, which results in less het-erogeneous flagellar glycosylation (373).

In contrast to the observations made in C. jejuni, the functionof flagellin glycosylation seems not to be involved in flagellar mo-tility or filament assembly in Pseudomonas (372). Also, the flagel-lar glycans are not the epitopes recognized by the human anti-type

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a flagellin monoclonal antibody (368). Nevertheless, flagellar gly-cosylation was shown to be involved in the virulence of Pseudomo-nas in a burn wound model (374) and the triggering of the inflam-matory response, as was shown by a comparison of IL-8production levels by A549 cells triggered by glycosylated and un-glycosylated flagellins (375). The exact in vivo role of flagellar gly-cosylation in Pseudomonas strains remains unclear.

The first Gram-positive organism for which flagellar glycosy-lation was reported is Listeria monocytogenes (376). The elucida-tion of flagellin glycosylation in L. monocytogenes resulted fromearlier reports on the discrepancy in molecular weight of its flagel-lin FlaA, which suggested a posttranslational modification (377,378). In 2004, the flagellins of listerial serotypes 1/2a, 1/2b, 1/2c,and 4b were shown to be �-O-glycosylated. Up to six sites of thecentral surface-exposed region of the flagellin monomer are gly-cosylated (376). The enzyme encoded by Lmo0688 (GmaR) waslater identified as a bifunctional GT protein that transcriptionallyregulates the expression of its enzymatic substrate, as it both har-bors �-O-GlcNAc-transferase activity and is an antirepressor ofthe MogR repressor, which represses flagellar production in atemperature-dependent manner. As Listeria species express theirflagella only at low temperatures, their expression is repressed at37°C (human body temperature) to evade recognition by the im-mune system (379). Based on mutational analyses of the GmaRGT, flagellar glycosylation in Listeria is believed to be unnecessaryfor the secretion, stability, and motility of the flagella, nor is itbelieved to be necessary for adhesion and biofilm formation (379,380). Further research will shed light on the functional impor-tance of flagellar glycosylation in this species.

Clostridium species are Gram-positive pathogens that targetthe GIT, and it has been speculated that their glycosylated flagellamight help them to establish an interaction with the host (381),although this remains to be documented. The first reports offlagellin glycosylation in Clostridium species were for C. tyrobu-tyricum and C. acetobutylicum ATCC 824 (382–384). C. botulinumwas the first human-pathogenic Clostridium species in which O-glycosylation of flagellin was studied. The genomes of these spe-cies harbor genes homologous to Leg biosynthetic genes of C. coli.The flagella are O-glycosylated with a new derivative of Leg on upto seven sites per monomer. Among C. botulinum isolates, a rangeof glycan structures was found (291). Strains targeting infants ap-pear to be glycosylated with Leg derivatives, while other strainsthat have no link to infections are generally decorated with hexu-ronic derivatives. This suggests a possible link between flagellinglycosylation and the pathogenic potential of Clostridium isolates.

The peritrichous flagella of Clostridium difficile strains and iso-lates, well-known GIT pathogens, are modified with O-linkedHexNAc residues. This modification seems to be conservedamong most strains. In C. difficile 630, up to seven sites of theflagellin (FliC) are modified with a HexNAc residue on which amethylated aspartic acid is linked via a phosphate bond. C. difficileclinical isolates have more heterogeneous glycans consisting of amaximum of five monosaccharides, again starting with a HexNAcresidue. In C. difficile 630, the GT CD0240 was further studied:inactivation of this gene led to a nonmotile phenotype, whichproduced only a limited amount of unglycosylated FliC. This find-ing illustrated the need for glycosylation of flagellin in the assem-bly and motility of flagella (381).

The bacterial agent causing syphilis, Treponema pallidum, ex-presses flagella composed of core proteins, FlaB, and sheath pro-

teins, FlaA. It was shown that the core protein, FlaB, is glycosylated(385). The nature of its flagellar glycosylation has yet to be furtherinvestigated, but roles for FlaB glycosylation in the regulation ofthe assembly of FlaA on the core flagellin and in the transport andstability of flagellar components were speculated.

Taken together, flagellar glycosylation has been reported formany species. In species harboring simple flagella, such as Pseu-domonas, glycosylation appears to be a nonessential feature, whilein species such as Campylobacter, Helicobacter, and Clostridiumspecies, glycosylation appears to be essential for the assembly andmotility of complex flagella. The latter fact seems to be typical forstrains colonizing the human GIT. Nevertheless, more studies areneeded to understand the functional importance of flagellin gly-cosylation.

Dedicated O-glycosylation of proteins: pili. Similar to glyco-sylated flagella, bacterial pili are often glycosylated. Some bacteriause a general system to do so, targeting both pili and other proteins(i.e., general O-glycosylation [see below]), and others harbor amechanism dedicated to pilus glycosylation.

We describe above the glycosylation of flagella in certain Pseu-domonas strains, while other strains express type IV pili, and somedecorate these pilins with glycans. The best-studied glycosylatedpilins are from P. aeruginosa 1244. Each PilA subunit of the pilusfiber is decorated with a single trisaccharide (386). The glycan isevenly distributed over the fiber surface, at the seam between twoadjacent pilins (285). The trisaccharide consists of 5-N-�-OHC47NfmPse, xylose, and FucNAc (386) linked to C-terminalserine 148 (387). This S148 residue lies adjacent to a disulfide loop,which is an important B-cell epitope (388). The terminal characterof pilin glycosylation is quite unusual, as proteins are mostly gly-cosylated on their core. Further comparison with a nonglycosylat-ing strain, PA103, resulted in the identification of this terminalserine as the major pilin glycosylation recognition feature. Thecompatibility of the surface also plays a role, as a positive charge ofthe adjacent disulfide loop appears to enable glycosylation, andthe terminal serine needs to be at a minimum distance from thedisulfide loop of the pilin subunit, probably to avoid steric hin-drance (389). These findings suggest that glycosylation occurs af-ter pilin folding, early in pilus biogenesis. Further investigation ofpilus glycosylation specificity revealed that only the structureof the sugar at the reducing end of the trisaccharide (FucNAc) is ofimportance, suggesting that the structural aspects of the remain-ing sugars are unimportant (390).

The trisaccharide made of 5-N-�-OHC47NfmPse, xylose, andFucNAc is similar to the O antigens of the LPS molecules of P.aeruginosa 1244, which suggests a common metabolic origin forboth oligosaccharides. Indeed, a mutant affected in O-antigensynthesis resulted in both the loss of O antigens and the loss ofpilin glycosylation (387). Heterologous expression of the O-anti-gen-producing machinery in E. coli further confirmed these find-ings. The use of the same pathway by P. aeruginosa 1244 to gener-ate glycans for LPS formation and pilin glycosylation is probablyan energy-saving process.

The structural pilA gene was found to be part of an operon alsoharboring pilO, which was shown to be required for pilin glycosy-lation (391). This O-OST has a sequence pattern that is also foundin WaaL (LPS ligase). The two enzymes compete for the samesubstrate, namely, lipid-linked monomeric O antigens, so it is nosurprise that they are part of the same Pfam Wzy_C family (51,392). Nevertheless, the active site of PilO differs from that of the



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other enzyme. The Wzy domain of PilO and the C-terminallyproximal hydrophilic portion of PilO are the regions linked toenzyme activity (392). PilO has a relaxed glycan specificity forshort oligosaccharides, which prevents the addition of polysac-charides that would impede pilus assembly (67). The factors de-termining this remarkable preference for short oligosaccharidesremain to be established. The only substrate specificity that couldbe found for PilO is the presence of a C-terminal serine or threo-nine residue and the above-mentioned minimum distance fromthe pilin surface and compatible surface charge (393).

PilO was shown to be the only pilin-specific glycosylation fac-tor (387, 391). Mutational analysis of PilO resulted in nonglyco-sylated pili that had a normal appearance but had somewhat re-duced twitching motility and were more sensitive to pilus-specificbacteriophages. Competition of this mutant with the wild-type1244 strain in a mouse model of acute pneumonia resulted ingreater survival of the strain carrying glycosylated pilins. This in-dicates that the glycosylation of pilins is an important virulencefactor that can interfere with the establishment of infection (285).These findings were in concordance with previous reports of thehigher frequency of group I Pseudomonas strains, such as 1244 andPAO1, in cystic fibrosis patients (394). There are five groups ofPseudomonas strains in total, which are defined based on DNAsequence comparisons of the pilin clusters (395). The glycans onthe pilin reduce the hydrophobicity of the pilin, which enablesthese strains to colonize other microenvironments (285).

Another P. aeruginosa strain harboring glycosylated pilins isstrain Pa5196 of group IV; later, glycosylated proteins were alsofound in strain PA7 (396, 397). These pilins are decorated with apeculiar homo-oligomer of �(1-5)-linked D-arabinofuranoses.This uncommon sugar also occurs in lipoglycans of mycobacteria.The D-arabinofuranose biosynthesis of Pa5196 is also highly ho-mologous to that of M. tuberculosis (398). The arabinofuranosesare present at several positions in the flexible central part of theprotein (397). The T glycosylation sites are modified with trisac-charides, and additional mono- and disaccharides can be foundon serine residues, which add up to about 16 arabinoses per pilinsubunit. The TfpW enzyme was identified as the pilin arabinosyl-transferase and is thus a potential O-OST (396), which pointstoward an en bloc glycosylation mechanism. The glycan substitu-tions are required for the efficient assembly of the pilus and inparticular for the establishment of subunit-subunit interactions(398).

A peculiar case of glycosylation of pili (or fimbriae) can befound in Streptococcus parasanguinis FW213, where the fimbria-associated protein Fap1, which was later identified as the majorconstituent of its fimbriae, is O-glycosylated (399, 400) (Fig. 8C).The Fap1 protein is a high-molecular-weight, serine-rich repeatprotein of 200 kDa and is decorated with Rha, Glc, Gal, GlcNAc,and GalNAc residues (400). The fimbriae of S. parasanguinis areimportant for adhesion to teeth, and the sugar residues present onFap1 mediate this process (400, 401). Glycosylation is essential forbiofilm formation of S. parasanguinis (401). The glycans on Fap1also protect the protein from degradation and stabilize its confor-mation (402).

Upstream of the fap1 gene is a biogenesis locus, comprising 3open reading frames (ORFs) (gap1 to gap3) and genes coding forputative GTs (gtf1, gtf2, gly, nss, galT1, and galT2) and SecA2 andSecY2 systems (403). The SecA2 system is an accessory secretionsystem (next to the canonical SecA pathway) and allows the trans-

port of glycosylated proteins, as glycosylation of Fap1 occurs in-tracellularly (402). The SecY2 protein not only forms the translo-cation channel but also is thought to play a role in the completeglycosylation of Fap1 (404). Also, the canonical SecA protein isthought to interact with the accessory SecA2 pathway (405). Thegap3 (orf3) gene of the locus upstream of fap1 plays a role in theglycosylation of Fap1 by linking it to the secretion of the protein.Gap3 is therefore of importance for fimbrial assembly, adhesion,and in vitro biofilm formation (406, 407). Gap1 also plays a role inthe biogenesis of Fap1 and mediates glycosylation. To do this, theprotein requires a conserved C-terminal 13-amino-acid motif(408). Taken together, Gap1 and Gap3 are accessory proteins thatinteract with SecA2 and mediate Fap1 biogenesis in this manner(SecA2-Gap1-Gap3 complex) (405) (Fig. 8C).

The Gtf1 GT catalyzes the first step in Fap1 glycosylation andadds GlcNAc (404, 408, 409). Gtf1 interacts with Gtf2 via its N-terminal domain (DUF1975), and the formation of this het-erodimer complex was proven to be required for optimal GlcNAc-transferase activity (409–411) (Fig. 8C). Gtf2 thus acts as amolecular chaperone via the stabilization, subcellular localization,and modulation of the activity of Gtf1 (411).

The second step in Fap1 glycosylation is carried out by theproduct of the nss gene, which was renamed Gtf3 (412) (Fig. 8C).The Gtf3 protein forms a new subfamily of GTs and shares homol-ogy only to other Streptococcus species harboring similar glycosy-lated serine-rich proteins (413).

The system described here is discussed further in the section onO-glycosylation of proteins in bacteria, below, as the associationof high-molecular-weight serine-rich repeat adhesin with an ac-cessory SecA2 locus and GTs is highly conserved in streptococcaland staphylococcal species (403).

In Streptococcus salivarius, K� and K� strains can be distin-guished, with the former having long fibrils and the latter havingfimbriae. Antigens B and C, present on the fibrils of K� cells, areglycoproteins (414), and the fimbriae of K� strains are assembledfrom glycoproteins that form a filamentous structure resistant todissociation (415).

Dedicated O-glycosylation of other proteins. The inability toglycosylate proteins of the most studied E. coli strains is widelyrecognized. E. coli is therefore extensively used as a vehicle for theheterologous expression of glycosylation systems to confirm theirability to produce glycosylated proteins and in glycoengineering.Nevertheless, some glycoproteins were reported for some, mainlypathogenic, E. coli strains. In 1968, an envelope-specific glycopro-tein was identified in E. coli strain B (416), followed in 1975 by areport of the glycosylation of the conjugative F pili (417). TheEDP208 and ColB2 F pili of E. coli K-12 were later further inves-tigated; the former were found to be unglycosylated, and the gly-cosylation status of the latter is still uncertain (418). Strikingly,these early reports of E. coli glycoproteins were not investigatedfurther to the best of our knowledge. In 2003, a Gp45 membraneglycoprotein containing mannose, glucose, galactose, and GalNAcresidues was identified in E. coli PCRO 1687 (419).

Better characterized are the self-associating autotransportersof E. coli, which are glycoproteins. Three autotransporters wereidentified in different strains: AIDA-I (adhesin involved in diffuseadhesion), TibA, and Ag43. All these autotransporters are impor-tant virulence factors modified with heptose residues that areadded by a single heptosyltransferase. These GTs are believed to be

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functionally interchangeable between strains, as they have similarspecificities (282, 420, 421).

A glycosylated TibA outer membrane protein was identified inenterotoxigenic E. coli (ETEC) strain H10407 (422, 423). Imme-diately upstream of the tibA gene, a tibC gene, coding for a hepto-syltransferase, was identified (423). The TibA protein is decoratedwith single, surface-exposed heptose residues, which is of impor-tance for the binding of ETEC H10407 to HCT8 cells, a humanileocecal epithelial cell line, in a specific and saturable manner.This suggests that TibA is an important factor in ETEC pathogen-esis, involved in adhesion and invasion (424). TibC homologueswere found in several but not all ETEC strains (420). In the sameETEC strain, H10407, an adhesin important for binding to intes-tinal epithelial cells, EtpA, was also found to be glycosylated by anadjacent enzyme (EtpC). Both genes, together with the etpB geneencoding a transporter, are part of the same locus (425).

The AIDA-I adhesin was identified in diarrheagenic E. coli clin-ical isolate 2787, and its glycosylation was found to be essential foradhesion to human intestinal cells (286). The main role of AIDA-Iis thought to lie in the stabilization of the protein at the cell sur-face, offering protection against the action of the numerous pro-teases present in the gut (282). As in TibA glycosylation, the hep-toses are added to AIDA-I by a heptosyltransferase, Aah (encodedby aidA), immediately upstream of the AIDA-I gene. AIDA-I ismodified with up to 19 heptose residues, which come from ADP-glycero-manno-heptose precursors from the LPS pathway of E.coli (286). Glycosylation was found to occur at the extracellulardomain, which is a �-helix and harbors imperfect repeats of 19amino acids. Further investigation of Aah specificity indicates thatthis enzyme recognizes a structural motif of �-helices rather thana specific sequence (426). Deletion of Aah resulted in a loss ofadhesion but retention of biofilm formation and autoaggregationcapacity.

The functional relationship between TibC and Aah was furtherinvestigated, and indeed, recombinant TibC can substitute forAIDA-I glycosylation (420). Furthermore, AIDA-I-specific anti-bodies can also recognize TibA. This strong homology suggeststhat Aah and TibC are members of a novel class of heptosyltrans-ferases, which transfer heptose residues to multiple sites on theprotein backbone (420).

A third autotransporter is the Ag43 protein, which is wide-spread in entero- and uropathogenic E. coli strains such asUTI536. Ag43 is also modified with heptoses (18 glycosylationsites were identified), but in contrast to TibC and Aah, no specificGT was identified upstream of the flu gene (421, 427). Its level ofhomology with TibA and AIDA-I is high, as Aah and TibC canglycosylate Ag43. Glycosylation was found to be important for theaffinity of Ag43 for human cells (421). Glycans also increased thestability of Ag43 against chemical and thermal denaturation andincreased refolding kinetics (427).

Several researchers reported the occurrence of glycoproteins inthe periodontal pathogen Porphyromonas gingivalis. In strainW50, two proteases, RgpA and RgpB, are glycosylated (428).These proteases are called Arg-gingipains and are extracellularcysteine proteases that degrade molecules such as collagen, fibrin,and fibronectin. RgpA occurs in three different forms: heterodi-meric RgpA (HRgpA), containing �-catalytic and �-adhesin do-mains, as well as monomeric RgpA and membrane-type RgpA(mt-RgpA), both harboring only the �-catalytic domain. Twopercent of the molecular weight of the heterodimer was attributed

to glycosylation, while this figure is 14% for monomeric RgpA andeven 30% for mt-RgpA. These percentages are similar for mono-meric RgpB, which can occur in a monomeric soluble form ormembrane-associated RgpB (428, 429). The glycan is linked to thecatalytic domain and cross-reacts with antibodies directed againstPorphyromonas LPS. This indicated that there are similarities be-tween the glycans present on the Rgp proteins and LPS structures(430). Later, similarity with special anionic cell surface polysac-charides (APSs) was also reported (431). In sera of adult patients,the glycans of RgpA are recognized as being immunogenic, whiledeglycosylation of the proteins abolishes this recognition (430).Glycosylation of the proteins also seems to modulate their stabil-ity, as the less glycosylated HRgpA has a shorter half-life than thatof RgpA (428). Aberrant glycosylation also had dramatic effects onthe stability of RgpA (429). RgpB was found to have an effect onmonomeric RgpA maturation (432). This is thought to occur viathe activation of the glycosylating enzyme or via the induction ofoligosaccharide extension (oligosaccharides of RgpA consist of 7to 35 sugar monomers) (429). The secretion, processing, anchor-age, and/or activity of the gingipains is modulated by the activityof the vimA and vimE gene products, where VimE is needed toestablish proper glycan biogenesis but is not a GT (433). Down-stream of vimE, a vimF gene encodes a putative GT, which altersthe glycosylation, anchorage, and activity of the gingipains (284).Details of this mechanism remain to be studied further.

Another reported glycoprotein in P. gingivalis is the highlyconserved outer membrane protein OMP85, the glycosylation ofwhich was shown to influence biofilm formation (434). The mi-nor fimbriae of 67 kDa (encoded by the mfa1 gene) were alsoshown to be glycosylated and target DC-SIGN (435). Via thisbinding, P. gingivalis can bind and invade DCs and elicit an im-munosuppressive response. A recently reported glycoprotein ishemin binding protein 35 (HBP35) (40 kDa), an outer membraneprotein involved in heme utilization. Like the arginine gingipains,HBP35 is secreted by the Por secretion system (436).

Aggregatibacter actinomycetemcomitans serotype b strainVT1169, an oropharyngeal pathogen, glycosylates a nonfimbrialextracellular matrix adhesin, EmaA. This protein trimerizes toform antenna-like structures that can bind collagen. EmaA ismodified with the O PS moiety of LPS containing fucose, rham-nose, and GalNAc (437, 438). Strikingly, the O PS is attached inthe periplasm to the protein via the activity of the O-antigen ligaseWaaL (437, 439). The glycan was proven to be crucial for collagenbinding and the protection of EmaA against proteolytic degrada-tion (439). Previously, the potential glycosylation of the Flp fim-briae was reported (440).

Two O-glycoproteins in the bacterium causing ehrlichiosis,Ehrlichia chaffeensis, were reported: P120 and P156 (441, 442).P120 is an immunodominant and surface-exposed protein mod-ified with glucose, galactose, and xylose residues attached to itsserine-rich tandem-repeat units (442).

Similar to the above-described Fap1 glycosylation in Strepto-coccus parasanguinis, other Streptococcus species and Staphylococ-cus species O-glycosylate their major serine-rich repeat adhesinvia accessory GTs, and secretion also occurs via an accessorySecA2 system (403).

Streptococcus gordonii M99 glycosylates GspB (280 kDa), a ser-ine-rich repeat protein (SRR1 and SRR2) important for adhesionto human platelets (443). Glycosylation occurs prior to export,and 70 to 100 monosaccharides are added, probably sequentially,



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to several sites on a GspB protein (10% of its MW), of which Glcand GlcNAc residues are the most predominant (444). Similarlyto Fap1 (Fig. 8C), a SecY2A2 locus can be found adjacent to thegspB gene, comprising accessory proteins (Asp1 to -5), SecY2,SecA2, and genes involved in GspB glycosylation (gtf, orf4, gly, andnss) (445, 446). The large glycoprotein is transported by theSecA2-SecY2 accessory system (443). Unglycosylated GspB canalso be transported by the canonical Sec system (447). Feltcherand Braunstein recently reviewed the accessory SecA2 system(448).

The accessory proteins are involved in the mediation of GspBsecretion (446). Asp2 and -3 bind the serine-rich repeats of GspBand are chaperones of the early phases of GspB transport (prior tofull glycosylation) (449). Findings suggest that Asp2 is a bifunc-tional protein, which also modulates, aside from transport, thedeposition of GlcNAc on the serine-rich repeats of GspB (450).Asp4 and Asp5 are functional homologues of the canonical SecApathway proteins SecE and SecG (451). Gtf and the protein en-coded by orf4 are required for the glycosylation and translation orstability of GspB and were renamed GtfA and GtfB, respectively(445). These proteins are homologous to the Gtf1 and Gtf2 initi-ating enzymes of S. parasanguinis and most probably perform thesame role in GspB glycosylation (411, 445). Gly adds the terminalsugars to GspB, and its N-terminal domain shares homology toknown GTs. Nss is thought to be involved in the biosynthesis ofUDP-sugar precursors (445, 446).

Strain DL1 of S. gordonii also expresses a serine-rich sialic acid-binding adhesin (like GspB), termed Hsa (452, 453). Both Hsa andGspB bind sialic moieties on the platelet membrane protein Ib�,the low-molecular-weight mucin MG2, and agglutinin present insaliva (454), showing that glycoproteins can also have lectin activ-ity themselves.

In S. sanguis, adhesion to platelets is also mediated by a serine-rich protein, SrpA, homologous to Hsa and GspB from S. gordonii(455). Other reported glycoproteins in this S. sanguis strain in-clude a fibrillar glycoprotein in strain 12 (456) and a platelet ag-gregation-associated protein (PAAP), which is thought to be N-glycosylated with rhamnose residues (281). This is the firstexample of N-glycosylation in Gram-positive species, althoughmore experiments are needed to confirm the N-glycosylation sta-tus of this protein.

A homologue of the Gtf1-Gtf2 system of S. parasanguinis and S.gordonii is also present in S. pneumoniae and glycosylates thepneumococcal serine-rich repeat protein PsrP (410, 457). Thisprotein also has serine-rich repeat domains and is accompaniedby the typical SecY2A2 locus (457).

A final well-studied example of a serine-rich glycosylated ad-hesin and its accessory locus can be found in Streptococcus agalac-tiae NEM316 (i.e., group B Streptococcus [GBS]), where Srr1 isglycosylated with GlcNAc and sialic acid residues prior to trans-port (458). GtfA and/or GtfB is essential for the production ofglycosylated Srr1, while the other accessory GTs are thought toplay a role in the adaptation of GBS in vivo (458).

The above-described conserved mechanism of glycosylationand transport of serine-rich repeat adhesins is also conserved in S.aureus strains. In S. aureus ISP479C, the SraP platelet-bindingvirulence factor is glycosylated (459). An accessory SecA2 locuscan be found, but Asp4, Asp5, Gly, and Nss are missing. SecY2,SecA2, and Asp1 to -3 are involved in the surface expression ofSraP, and the GTs GtfA and -B modulate its glycosylation (460).

Of 21 isolates investigated by Siboo and coworkers, 85% wereshown to express SraP (459).

The 60-kDa immunodominant glycoprotein IDG-60 (generalstress protein GSP-781) of S. mutans is modified with sialic acid,Man, Gal, and GalNAc residues. This glycoprotein is essential forthe maintenance of cell wall integrity and cell shape and, thus, forbacterial survival under stress conditions (461).

Streptococcus faecium ATCC 9790 expresses a glycoenzyme thathas autolytic N-acetylmuramoylhydrolase activity and is deco-rated with O-linked monomers and oligomers of glucose (91).Also, glycosylation of a PG hydrolase in L. rhamnosus GG, Lacto-bacillus plantarum WCFS1, and Lactococcus lactis MG1363 wasreported (89, 90, 92). For Lactobacillus buchneri strains CD034and NRRL, the glucosylation of putative glycosylhydrolases(LbGH25B and LbGH25N, respectively) was reported recently(93).

The MspI (p75) protein of L. rhamnosus GG is O-glycosylatedwith concanavalin A (ConA)-reactive sugars (89). Its glycosyla-tion was shown to have an impact on the stability of this PGhydrolase and protect against proteases. However, glycosylationwas not essential for the PG-hydrolyzing activity and Akt signalingcapacity of MspI (89). Acm2, the major autolysin of L. plantarumWCFS-1, is intracellularly glycosylated on its N-terminal AST do-main, a domain that is rich in A, S, and T residues (90). Glycosy-lation was recently shown to be an autoregulating factor of theenzyme and to have a negative impact on its N-acetylglucosamini-dase activity. Twenty-one mono-GlcNAc substitutions werefound in the AST domain of Acm2, which help to stabilize theenzyme against proteases (90, 462). A broader screening of theglycoproteome of L. plantarum WCFS1 revealed 10 novel glyco-proteins: 5 that have an intracellular nature, 2 extracellular PGhydrolases, and 1 mucus-binding protein. Most glycoproteins aremodified with 1 HexNAc residue, but in some cases, hexose moi-eties were detected (463). Based on homology with the Gtf1 GT ofFap1 of S. parasanguinis (see above), the GtfA and GtfB GTs re-sponsible for the O-glycosylation of Acm2 were recently eluci-dated (464). Taken together, these findings point toward a generalmechanism of glycosylation in this species, but this remains to befurther substantiated.

General O-glycosylation systems. A few bacterial species con-tain a glycosylation system that targets a diverse set of proteins,often including pili and adhesins. In such “general systems,” thegenes encoding the protein substrates are not clustered with thegenes encoding the GTs and transport functions, in contrast todedicated glycosylation systems.

(i) General O-glycosylation of neisserial proteins. The best-studied general O-glycosylation mechanisms are found in Neisse-ria gonorrhoeae, or gonococcus, and in N. meningitidis, alsoknown as meningococcus (Fig. 8D). Both species are importantGram-negative human pathogens, the former causing gonorrheaand the latter causing meningitis.

In 1977, the first report on gonococcal pilus glycosylation(strain P9) was published (465). Dedicated research on the glyco-sylation of meningococcal and gonococcal pili emerged only inthe early 1990s (289, 466–470) and has remained a hot topic inbacterial glycosylation with the discovery of the general characterof the glycosylation machinery in 2009 (471).

N. gonorrhoeae strain MS II cells are covered with type IV pili,and the S63 residue of this pilin subunit (PilE) is modified with acovalently bound O-linked GlcNAc-�(1-3)Gal dimer (472). The

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formation of the glycosidic bond between Gal and GlcNAc is cat-alyzed by a pilin galactosyltransferase, PgtA. In more than half ofthe clinical N. gonorrhoeae isolates tested by Banerjee and cowork-ers (40 out of 70), this gene contains a poly(G) tract, which rendersPgtA prone to phase variation (473). Remarkably, the poly(G)tract was present in all clinical isolates of patients with dissemi-nated gonococcal infection (24 out of 24 isolates tested). Thephase variation of PgtA is thought to be involved in the conversionof uncomplicated gonorrhea to a disseminated gonococcal infec-tion, suggesting a clear link between PgtA and the glycosylated piliof N. gonorrhoeae as virulence factors (473). The phase variation ofgonococcal pilus glycosylation appears to enhance the immuneevasion properties of the pilin glycans and, thus, the survival of N.gonorrhoeae in human blood (472, 474).

In another N. gonorrhoeae strain, i.e., N400, pilin is glycosy-lated on S63 with a hexose linked to a proximal 2,4-diacetamido-2,4,6-trideoxyhexose (HexDATDH) (475), with DATDH beingdiNAcBac (di-N-acylated bacillosamine) (476). Other glyco-forms, such as DATDH, HexHexDATDH, and O-acylated formsof DATDH linked to hexoses, are also found, which rounds up toa total of five different glycoforms in this N400 strain. The glycanis transferred en bloc to its substrate after assemblage on a lipidcarrier (477) (Fig. 8D). Glycan biosynthesis is distantly related tothe N-glycosylation pathway of C. jejuni (476). DATDH is theproduct of the priming GT PglB, a bifunctional acetyltransferaseand phosphoglycosyltransferase characterized mainly in N. men-ingitidis strains (478). In some strains and isolates, PlgB is replacedby PglB2, resulting in GATDH (2-acetamido-4-glyceramido-2,4,6-trideoxyhexose) sugar moieties (479). PglC was identified asa transaminase, PglD was identified as a dehydratase, PglF wasidentified as a flippase, and PgtA was renamed PglA. PglA is a GTthat transfers the hexose moiety, i.e., galactose, to DATDH. PglE isa GT involved in the transfer of the second hexose residue toHexDATDH. Both PglA and PglE are prone to phase variation,which results in the mono-, di-, and trisaccharide glycoforms.Moreover, PglE shows promiscuity toward Hex and HexNAc res-idues. The OST PglO shows a relaxed specificity toward mono-,di-, and trisaccharides, and mutation of this enzyme has beenshown to result in the absence of glycan modifications (477). Theglycan can be O-acetylated by the activity of the PglI acyltrans-ferase enzyme, which uses acetyl coenzyme A (acetyl-CoA) as adonor (477). Of interest, many N. gonorrhoeae and N. meningitidisstrains were found to harbor another GT, PglH, which has a func-tion similar to that of PglA. As this would result in a metabolicconflict with PglA, a conserved deletion inactivates PglH in moststrains. This results in reduced glycan diversity, as the action ofPglH results in unique disaccharide products modified with glu-cose residues instead of galactose (480, 481). Taken together,PglD, -C, and -B synthesize the UDP-sugars, and PglB, -A, -E, and-H form the core locus of glycan assembly, while the PglO OSTtransfers the finished glycan en bloc to the substrate (476, 480)(Fig. 8D).

N. gonorrhoeae strains can thus modulate their glycan diversityto enhance their virulence by the expression of a range of glyco-forms resulting from their ability to express mono-, di-, and tri-saccharides; use both GATDH (PglB2) and DATDH (PglB); po-tentially acetylate (PglI) sugars; and exploit PlgH and the phasevariability of PglA and PglE (476).

In 2009, the en bloc glycosylation mechanism of gonococcalpilins was shown to be a general system also targeting several

membrane-associated proteins in addition to pili (471). Elevenglycoproteins were identified, sharing an S-A-P-A motif that wasnot sufficient or necessary for glycosylation. The glycoproteinsinclude the disulfide oxidoreductase DsbA, various proteins in-volved in electron transport systems and redox components, andproteins involved in solute uptake. These findings imply the pro-miscuous character of PglO, having various substrates (471). In2012, another six new glycoproteins were reported, with PilQ, asecretin critical for the ability of pili to reach the cell surface, beingthe most important example (482, 483).

Not only was the PilE pilin shown to be glycosylated, it is alsomodified with phosphocholine and phosphoethanolamine resi-dues on Ser68 (475). The clustering of both modifications on pilinpeptides results in a dynamic interplay and even direct substratecompetition between phosphoform modifications and O-linkedglycans (484).

Functionally, the glycan chain length modulates differentialeffects on gonococcal growth, while glycoform variation affectsthe intrinsic processes of pilus dynamics and interactions betweensubunits (485). Also, the glycosylation of pilin has been linked tothe activation of complement receptor 3 (CR3) of primary cervicalepithelial cells by N. gonorrhoeae 1291 (486) and adhesion of N.gonorrhoeae F62 (487). Neisseria strains typically also producetruncated soluble pilins, so-called S-pilins, of unknown function(467). These S-pilins were found to be more abundant in gono-coccal strains, and glycosylation of the Ser63 residue further in-creased, but was not necessary for, S-pilin production. Thesefindings are in contrast to those for N. meningitidis, where glyco-sylation of Ser63 is crucial for the production of S-pilins. S-pilinproduction is thus divergent in both species (467).

The general O-glycosylation mechanism of N. meningitidis isalso well studied and similar to the one in N. gonorrhoeae. In 2009,Ku and coworkers demonstrated that the pilin glycosylationmechanism of N. meningitidis is also a general system (Fig. 8D)with the discovery of the C-terminal glycosylation of the nitritereductase AniA in strain C311 (488).

Pilin glycosylation in N. meningitidis was first noted by thediscrepancy in molecular weights of the pilins of N. meningitidisstrains C311 and MC58 (489). Further investigation of the PilEpilin of strain C311 revealed an O-linked trisaccharide consistingof a terminal �(1-4)-linked diGal covalently linked in an �(1-3)manner to a DATDH sugar, which is directly attached to the pilin(468, 470), likely on the S63 residue (289). Class I N. meningitidisstrains show O-glycosylation of S63 with the Gal �(1-3)GlcNAcdisaccharide and are highly similar to some gonococcal glycosy-lated pili (289, 467). Pilus glycosylation is variable between me-ningococcal strains and isolates, but most strains have covalentlylinked galactose residues modifying their pili (490).

In N. meningitidis strain MC58, PglA was identified as thepilin-specific galactosyltransferase responsible for the formationof the �(1-3) linkage between galactose and DATDH (466). PglAalso shows substrate flexibility toward both Gal �(1-3) DATDH(N. meningitidis strain C311) and GalGlcNAc (N. gonorrhoeaeand, e.g., N. meningitidis strain 8013SB) (491). Mutation of PglAhad no effect on pilus-mediated adhesion to human epithelial andendothelial cells. This enzyme has a homopolymeric G tract and isthus phase variable (466). PglA of N. gonorrhoeae and N. menin-gitidis catalyze similar linkages using distinct substrates and arehighly homologous (96% sequence identity). The N. gonorrhoeaeenzyme can be constitutively expressed or shows phase variability



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in some cases; the meningococcal enzyme, in contrast, is alwaysphase variable (473). As mentioned above, N. meningitidis strainscan also express PglH, a GT for which the activity results in uniqueglycans but which is inactive in most strains (480, 481).

PglE in strain C311 was identified as a galactosyltransferase,catalyzing the transfer of the terminal galactose. The gene encod-ing this enzyme has up to 24 copies of a heptant repeat, whichresults in phase variation between di- and trisaccharide structures.The number of heptant repeats varies between different strains(492).

Also in C311, the clustered PglB, -C, and -D enzymes wereidentified as being involved in the biosynthesis of DATDH, withPglB being the GT that transfers the sugar from the activated nu-cleotide directly to the lipid carrier. PglB was found to be bifunc-tional, as it also acetylates C-2 or C-4 of the diacetamido sugar.The PglC aminotransferase transaminates C-2 or C-4, and PglD isa dehydratase acting on C-2, C-4, or C-6 (478). PglB, -C, -D, and-A are present in all Neisseria species, which suggests a commonpilus glycosylation mechanism (466, 478). PglI was also found toplay a role in DATDH biosynthesis and shows homology to acety-lases (491).

After assembly of the glycan on the lipid carrier, the UndP-linked trisaccharide intermediate is transferred by the PglF flip-pase to a position where the glycan can be transferred to the pilinsubunit prior to pilin polymerization (479, 493). In particular, thepyrophosphate linker between the glycan and the carrier needs tobe adjacent to the OST (66). The PglF enzyme is similar to theWaaL enzyme involved in E. coli O-antigen biosynthesis (493).The involvement of a lipid-linked oligosaccharide intermediate inthe glycosylation process was confirmed by using mutants with ablockage in the pilin glycosylation pathway; these mutations alsointerfered with capsular transport and assembly (479).

After translocation of the lipid-linked oligosaccharide into theperiplasm, the PglL OST transfers the glycan to the substrate (Fig.8D). Expression of PglL, pilin, and diverse UndP-linked glycans ofE. coli strain MC58 resulted in glycosylated pilins, which provedthe relaxed specificity of PglL and its capability for the transfer ofdiverse oligo- and polysaccharides. The only requirement seemsto be the translocation of the saccharides into the periplasm (67).PglL shows 95% identity to the gonococcal OST PglL (67) and isalso similar to the O-antigen ligase of E. coli (493). As mentionedabove, these O-OSTs represent a novel family, the members ofwhich share a homologous domain of 30 amino acids, which isalso present in the WaaL LPS enzyme (67).

The promiscuity of PglL was further tested and resulted in thefinding that PglL can transfer virtually any glycan from a UndPcarrier to pilin in both E. coli and Salmonella (66). PglL even in-terfered with peptidoglycan biosynthesis (66). It was thought thatthe substrate specificity of PglL lies primarily in the lipid carrier. Inparticular, PglL recognizes C-1 and O-1 of the monosaccharide atthe reducing end of the glycan, the pyrophosphate linker, and thefirst few carbon atoms of the lipid carrier (66). Later studiesshowed that the catalytic efficiency of PglL is linked to the lengthand conformation of the acyl chain of the glycan donor. PglL caneven act as a Leloir enzyme and transfer sugar moieties directlyfrom activated nucleotide donors. This means that the lipid carri-ers influence glycosylation but are not essential for PglL function(68). Taken together, these results prove the extreme promiscuityof PglL, which opens new avenues in glycoengineering.

Nearly half of clinical isolates have an insertion in the pglBCD

operon, particularly in pglB, which results in different glycosyla-tion of the pili (492). This insertion results in a different form ofPglB, called PglB2, which is a glyceramidotransferase (494). PglB2has a functionally distinct C terminus, resulting in a differentmodification of the acetamido sugar (479). The net result is aGATDH sugar instead of DATDH (494). Moreover, PglB2 can beexpressed by two different alleles, which results in extra glycandiversity (495).

As in N. gonorrhoeae, the glycosylation of pilin is susceptible tovariation, both by the gene complement expressed by strains andby the phase variability of the genes present (492). For example,PglE and -F are present in all strains studied, in contrast to PglG,-H, and -B2, which are not present in C311 but are expressed in alarge number of clinical isolates. Moreover, PglG, -H, and -B2 arepotentially phase variable (481, 492, 495). Together with the ex-treme promiscuity of the PglL OST, this results in an enormousglycan repertoire, which confers an evolutionary advantage to N.meningitidis (66, 496). In analogy to N. gonorrhoeae, it was hy-pothesized that this glycan modification serves as a cloak for hostimmune responses (66, 289, 290). This is illustrated by the bindingof anti-Gal antibodies to the pilin of N. meningitidis, which resultsin the blockage of complement-mediated lysis of the cells (290).The diversification of the glycans present in N. meningitidis chal-lenges selection by the innate and adaptive immune system of thehost (496). The pili of N. meningitidis are structurally variable andinvolved in adhesion processes (469, 489). Nevertheless, glycanmodification of the pili appears to play no major role in piliationand adhesion but facilitates the solubility of pilin monomers (289,492).

(ii) General protein O-glycosylation in Bacteroidetes. Anotherimportant example of general O-glycosylation of proteins in bac-teria can be found in the order Bacteroidales, where these systemswere found across the entire Bacteroidetes phylum (497), Flavo-bacterium meningosepticum (498), and Tannerella forsythia (499).

B. fragilis is a common inhabitant of the human colon (10 to20% of the colon microbiota) and uses a mammalian-like path-way to decorate numerous surface capsular polysaccharides andglycoproteins with L-fucose (139, 270). Fucose is an abundantsurface modification of intestinal epithelial cells, and the coordi-nated expression of similar surface molecules by the colonic sym-biont and its host, i.e., molecular mimicry, results in a competitivecolonization advantage for the B. fragilis symbiont (139). It wasfound that microorganisms, and B. fragilis in particular, evenstimulate the terminal fucosylation of glycoproteins and glycolip-ids of intestinal epithelial cells (500). B. fragilis strain 9343 cancleave these L-fucose residues to internalize them and use them asan energy source. More strikingly, this strain can convert theseexogenously acquired sugars to GDP-L-fucose for incorporationinto its own surface capsular polysaccharide structures and glyco-proteins (139).

In 2009, a general O-glycosylation system in B. fragilis 9343,which glycosylates proteins with roles in chaperone-requiringprocesses (BF0994 and BF0447), protein-protein interactions(BF2334 and BF2494), and peptide degradation (BF0935 andBF3918) and surface lipoproteins (BF0522 and BF3567), wascharacterized. The eight identified glycoproteins were all presentin the periplasm or outer membrane and shared a common three-residue motif, which was later identified as D-(S/T)-(M/I/A/L/T/V). These residues are thought to be involved in both the catalysisof O-glycosylation and recognition (269, 270). Engineering of this

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motif in naturally unglycosylated proteins resulted in site-specificglycosylation, which proves the glycoengineering potential of thisstrain (269).

An in silico screening of the proteome of B. fragilis based on thepresence of this glycosylation motif and the fact that a signal pep-tidase I or II cleavage site or transmembrane-spanning domainshould be present (periplasmic and outer membrane localizationsof eight previously confirmed glycoproteins) predicted 1,021 can-didate glycoproteins. Twelve of these glycoproteins were con-firmed experimentally, and four of them were predicted to localizeat the inner membrane. Remarkably, four of the confirmed glyco-proteins have roles in cell division and chromosomal segregation(BF0066, BF0252, BF0289, and BF0586), two of which are en-coded by essential genes (FtsI [BF0252] and FtsQ [BF0259])(269). This may point toward a role for protein glycosylation indivisome regulation, which is in accordance with predicted glyco-proteins of L. rhamnosus GG and C. jejuni (569). Later, the num-ber of essential candidate glycoproteins was augmented to 43(497).

The confirmation of the glycosylation status of all 12 potentialextracytoplasmic glycoproteins carrying the glycosylation motifmay imply the presence of hundreds of extracytoplasmic glyco-proteins, which would account for more than half of the totalproteins present in the extracytoplasmic fraction, i.e., 365 out of762 proteins (269).

The glycan added to the proteins is an oligosaccharide of nineunits, which contains a fucose and is attached via a hexose (e.g.,mannose) to the protein substrate (501). Further investigation ofthe glycan revealed the presence of a core and an outer glycan,which are independently built. The genetic region (lfg gene locus[see below]) encoding proteins involved in the biosynthesis of theouter glycan is conserved within species but divergent betweenspecies, while the genetic region for the core glycan is more con-served among species (core glycan is absent in a gmd-fcl fkpstrain) (497). The enzymes (GTs and Wzx flippase) responsiblefor the en bloc glycosylation of proteins are clustered in the lfg genelocus. Strikingly, despite the high level of homology between O-OSTs, no OST could yet be uncovered (270). The transcription ofthis locus is linked to metG (encoding a methionyl-tRNA synthe-tase) transcription, which links protein synthesis and glycosyla-tion (497).

This general en bloc O-glycosylation system is central to thephysiology of Bacteroides, as a substantial number of proteins indiverse locations and with a range of biochemical functions werefound to be glycosylated. Moreover, no unglycosylated forms ofthese proteins could be recovered. Glycosylation seems to be vitalfor protein synthesis, and deletion of the glycosylation machinerywas shown to result in a strain deficient in growth and unable tocompetitively colonize the GIT (270, 497). It is likely that thismachinery confers a competitive colonization advantage to thesespecies (139, 269). Functionally, the glycosylation of proteins isthought to confer labels for binding and recognition and improvethe stability, solubility, and resistance of proteins to proteases(270).

The glycosylation machinery is conserved among Bacteroidesspecies (270) and even phylum wide: the four diverse classes ofBacteroidetes also harbor the O-glycosylation machinery, i.e., Bac-teroidia (B. fragilis), Flavobacteria (F. meningosepticum), Sphingo-bacteria, and Cytophaga (T. forsythia) (497, 498, 501). This pointstoward the early emergence of the general O-glycosylation system

in evolution, before the divergence of the four Bacteroidetesclasses. It is believed that this mechanism is maintained in view ofits physiological importance to diverse species of the phylum(497).

Both B. fragilis and T. forsythia use an identical glycosylationmotif, which allows the heterologous expression of glycoproteinsof one species in the other (501). T. forsythia is a periodontalpathogen covered with an S-layer built up by two glycoproteinsthat show no homology to other known S-layer (glyco)proteins:TfsA and TfsB (502). Later, other glycoproteins, such as the BspAsurface antigen, were also discovered (499, 503). The oligosaccha-ride modifying the glycoproteins of T. forsythia contains mannose,L-fucose, and Pse residues and is the result of genes in a 6- to 8-kbconserved locus (TF2049 to TF2055) (499). The S-layer of T. for-sythia was identified as being an important virulence factor, as ithelps the pathogen evade recognition by the innate immune sys-tem of the host (504, 505).

The 3-amino-acid glycosylation motif was also conserved inFlavobacterium meningosepticum, where several secreted proteinswere found to be glycosylated at a D-S or D-T-T motif with apeculiar 7-residue oligosaccharide (498, 506).

(iii) Francisella tularensis. F. tularensis is a pathogen thatcauses tularemia, a severe, potentially fatal zoonotic disease, and istherefore classified as a potential bioterroristic agent. The bacte-rium expresses a type IV pilin, PilA, which appears to be glycosy-lated based on aberrant migration on SDS-PAGE gels (507). Ex-pression of PilA in N. gonorrhoeae resulted in glycosylated pilins(508). Later research confirmed pilin modification with a pentasa-ccharide consisting of hexoses and acylated hexoses. Glycoformsof the pentasaccharide were also found, carrying unusual moietieslinked to the distal sugar via a phosphate bridge (509). Apart fromthe pilin, other proteins linked to virulence were also found to beglycosylated. Balonova and coworkers used a combination oftechniques (periodic acid-Schiff base stain, lectin blots, and lectinaffinity chromatography) to identify 15 new candidate glycopro-teins, including a protein involved in cell division (FtsZ[FTSH_1830]); a thioredoxin family protein, DsbA (FTH_1071);an OmpA family protein, FTH_0323; and several chaperones(GroEL [FTH_1651] and DnaK [FTH_1167]) (510). Of note,many of these proteins were also identified by Janovska et al. (511)as immunoreactive antigens in the live vaccine strain (LVS) of F.tularensis subsp. holarctica reacting with human tularemic sera.Interestingly, the glycosylated lipoproteins FTH_1071 andFTH_0414 were also previously found to interact with TLR-2/TLR-1 heterodimers of HeLa cell lines, resulting in the inductionof a proinflammatory response (512). Since interaction with theseTLRs is generally mediated via acylation, glycosylation is thoughtto play a modulatory role, as shown by Sieling et al. (513). Thoseresearchers demonstrated that glycosylation of the mycobacteriallipoglycoprotein LprG is required for the stimulation of innateimmune responses via activation of major histocompatibilitycomplex (MHC) class II-restricted T cells. The DsbA protein(FTH_1071), an essential virulence factor of Francisella, is modi-fied with a hexasaccharide, which seems to be nonessential forvirulence in vivo in a murine model of tularemia (514). Anotherimportant immunoreactive virulence factor, encoded by FTH_0069,is glycosylated with the same hexasaccharide as that of DsbA andPilA. This oligosaccharide consists of two hexoses, three N-acetyl-hexosamines, and an unknown monosaccharide containing aphosphate group (515).



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A gene cluster (FTT0789-FTT0800) was identified in strainFSC200, which contains three GT homologues, a GalE homologue(FTT0791), a transporter, and three hypothetical proteins (514).This cluster plays a role in glycan biosynthesis, and the FTT0791and FTT0798 gene products are implicated in DsbA glycosylation,with FTT0791 being the GalE homologue and thus a putativeUDP-Gal-4-epimerase (514).

The OST involved in the en bloc glycosylation of the proteinsubstrates was also identified: PglA, encoded by FTT0905, inFSC200. The expression of PglA is modulated by MglA, a viru-lence regulator also required for pilus expression (509, 516). PglAis conserved in Francisella species, and expression of PglA and PilAin E. coli proved to be sufficient for pilin glycosylation (509). Ho-mologues of PilA and PglA are found in all strains of the Franci-sella genus, and the glycosylation machinery seems to be a generalfeature of these strains (509).

(iv) Acinetobacter baumannii. Very recently, a general O-linkedprotein glycosylation system was discovered in the “superbug” A.baumannii, known as a causative agent of nosocomial infections.In A. baumannii ATCC 17978, seven glycoproteins were identi-fied, including the OmpA protein A1S_1193, each modified with abranched pentasaccharide of GalNAc, Glc, Gal, GlcNAc, and atriacetylated glucuronic acid (517).

Strikingly, a common pathway synthesizes this pentasaccha-ride and the capsular polysaccharides. PglC (A1S_0061), thepriming GT, initiates the production of the pentasaccharide viathe transfer of a GlcNAc residue to the lipid carrier. Further as-sembly is carried out by four GTs, encoded by A1S_0058,A1S_3482, A1S_0059, and A1S_0060. Once this oligosaccharide isfinished, Wzx (A1S_0056) flips the structure to the periplasm.This pentasaccharide modifies glycoproteins and also serves as abuilding block for the capsular polysaccharides. The pathways di-verge in the periplasm: glycoproteins are further processed by thePglL OST, and capsular polysaccharides are polymerized by Wzy(A1S_3483) (518). The PglL OST involved in the biosynthesis ofthe glycans is encoded by A1S_3176 and shows homology to theother O-OSTs (517). Furthermore, the maximum glycan modifi-cation found on glycoproteins is a decasaccharide, suggesting theinability of PglL to access polymerized glycans and use them as asubstrate (518).

Abolishment of the glycosylation machinery in A. baumanniiATCC 17978 resulted in diminished biofilm formation, reducedvirulence in two infection models (Dictyostelium discoideum andlarvae of Galleria mellonella), and reduced in vivo fitness in amouse model of peritoneal sepsis (517, 518). Both glycoproteinsand capsular polysaccharides are important virulence factors of A.baumannii for infection in animal models.

Despite the genome plasticity of A. baumannii strains, the O-glycosylation machinery is present in all clinical isolates and allsequenced genomes. This indicates a strong evolutionary pressureto maintain this feature (517).

(v) Peculiar glycoproteins in Actinomycetes. A peculiar generalO-glycosylation mechanism is found in Actinomycetes, of whichM. tuberculosis, Mycobacterium leprae, and Mycobacterium bovisare important human pathogens. These species decorate theirproteins with mannose residues following an evolutionarily con-served mechanism similar to eukaryotic O-mannosylation pro-cesses (519). The identification of glycoproteins in these actino-mycetes was based mainly on their reaction with the ConA lectin,which specifically binds to mannose residues (520–522). Most of

the identified glycoproteins are also acylated. In 1989, Espitia andMancilla identified a glycoprotein of 38 kDa and a doublet of 50 to55 kDa in Mycobacterium tuberculosis H37Rv (520). The glycosy-lation of the doublet was later confirmed by digestion of the gly-cans with the �-mannosidase from jack bean (523). The 38-kDaglycoprotein is involved in a phosphate-specific transport systemand is better known as PstS1 or PhoS1 (Rv0934) (524, 525).

A 19-kDa major membrane lipoprotein and known virulencefactor, LpqH (Rv3763), was also found to be ConA reactive (522,525), and its glycan substitutions seem to regulate the cleavage ofa proteolytically sensitive linker region (526). The glycan thusconfers resistance to proteases to LpqH and in doing so retains theprotein on the cell. Cleavage of the linker, in contrast, results in anonacylated secreted antigen (526). LpqH is a major adhesin andTLR-2 agonist and was found to bind to mannose receptors ofTHP-1 monocytes. In doing so, LpqH was shown to promote thephagocytosis of mycobacteria (527). Another adhesin, HBHA(heparin-binding hemagglutinin), is also glycosylated, and theglycan residues are thought to protect the lysine-rich C terminusof the protein against proteolytic attack (528).

Apa (MPT32), encoded by Rv1860, is a ConA-reactive glyco-protein of 45 kDa that is rich in proline. Four percent of its mo-lecular weight can be attributed to the presence of glycan moieties(521). Further research showed the presence of 5 glycopeptides inwhich a T residue is modified with a mannose, �(1-2)-linkedmannobiose, or �(1-2)-linked mannotriose (529). The most com-mon glycoforms are modified with 6, 7, or 8 mannose residues;Apa proteins modified with 3, 4, or 5 mannoses are also found, butglycoforms harboring 0, 1, 2, or 9 mannoses are rare (288).

Recombinant expression of Apa in M. smegmatis and a non-glycosylating E. coli host resulted in a decreased capacity to stim-ulate T-lymphocyte responses in guinea pigs (287). Moreover,deglycosylation of Apa generally decreased the capacity of the pro-tein to elicit cellular immune responses in vivo and in vitro. Degly-cosylated Apa is 10-fold less active in delayed-type hypersensitiv-ity reactions in immunized guinea pigs and 30-fold less active inthe stimulation of T lymphocytes in vivo (288). These studies in-dicate that mannose is an essential component of Apa as an anti-gen and in the modulation of T-cell-dependent immune re-sponses (287, 288). Lara et al. showed that tuberculosis patientscarry antibodies that react mainly to the carbohydrate part of Apa(530). M. tuberculosis targets the pulmonary surfactant protein A(PSP-A) C-type lectin of the innate immune system mainly via itslipoglycans, but the Apa antigenic glycoprotein was also suggestedto aid in attachment to this receptor (531).

Most of the glycoproteins mentioned here are T-cell antigens,but the SodC superoxide dismutase (Rv0432), a B-cell antigen,was also found to be glycosylated at six residues in its N terminus.Strikingly, apart from glycans attached to threonine residues, gly-cosylated serine residues were also identified, which represents thefirst evidence of serine-linked O-glycosylation in Mycobacteriumspecies (532).

In view of the evolutionary relatedness to O-mannosylation inSaccharomyces cerevisiae, mechanistic studies on O-protein glyco-sylation in M. tuberculosis were performed and resulted in theidentification of an O-mannosyltransferase encoded by Rv1002c,which initiates protein mannosylation (519). This enzyme is amembrane protein, and Sec translocation of the protein is re-quired for its mannosylation, which also implies the need for alipid carrier for the glycan residues (519). The Rv1002c enzyme is

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crucial to the virulence of M. tuberculosis, as mutation of the en-zyme, and the subsequent loss of mannoproteins, severely impairsin vitro growth and attenuates the virulence of M. tuberculosis(533).

Some glycosylated antigens were identified in the closely re-lated species M. bovis, a pathogen targeting mainly oxen but alsohumans (534). The MPB83 antigen is the best-characterized oneso far and is modified at two threonine sites with mannose and�(1-3) mannobiose. This is peculiar, as the MPT32 mannose res-idues of M. tuberculosis are �(1-2) linked (535).

For M. leprae, no protein glycosylation mechanism has beendescribed yet, but the LprG lipoprotein was found to be glycosy-lated and to activate TLR-2 via its carbohydrate moiety, which isstriking, since normally, acyl chains interact with TLR-2. More-over, the glycan is required for the inhibition of IFN-�-inducedMHC class II T-cell activation and antigen presentation by mono-cytes to T cells by TLR-2-dependent mechanisms. A homologue ofLprG is also present in M. tuberculosis (Rv1411c), and a homo-logue of Rv1002c was found in the genome of M. leprae (513).

(vi) Indications of general O-glycosylation systems in otherspecies. A conceptual study by Gebhart and coworkers revealedthe presence of O-OSTs in Vibrio cholerae N16961 (VC0393) andBurkholderia thailandensis E264 (BTH_I0650) (536). To the bestof our knowledge, no glycoproteins have yet been reported for V.cholerae, but it was shown that it harbors the potential to express aglycosylation system in its genome (536). It remains to be estab-lished if this system is functional in a native background.

In the species Burkholderia thailandensis and B. mallei, flagellarO-glycosylation was reported previously. In B. pseudomallei, agene of the LPS O-antigen cluster (rmlB) was found to be involvedin flagellar glycosylation (537). Recently, it was hypothesized thatthe BimA autotransporter of B. mallei might be glycosylated byBimC (538). Taken together, Burkholderia species possess thegenomic content to carry out protein glycosylation, but the natureof this system remains to be elucidated.

It is also not inconceivable that more general protein glycosy-lation systems have yet to be identified. The above-describeddedicated systems targeting flagella, pili, and other proteins couldtarget more proteins, since the elucidation of the general O-glyco-sylation system of Neisseria species was also initiated by the dis-covery of glycosylated pili (see above). The discovery of multipleglycoproteins in L. plantarum is an example of a bacterium inwhich a general mechanism is very likely to be found (463) (seeabove). Moreover, the protein glycosylation potential of a range ofbacteria has not yet been studied.

S-glycosylation of proteins. The modification of cysteine resi-dues with glycans occurs regularly in eukaryotes. This phenome-non was reported only recently for a bacterium. Lactobacillus plan-tarum KW30 produces a bacteriocin, glycocin F, which isglycosylated on a serine with a GlcNAc residue and also on a cys-teine residue with a HexNAc (272, 273). Serine-linked glycosyla-tion was found to be essential for the bacteriostatic activity of thebacteriocin, while the cysteine-linked glycan enhances bacterios-tasis. The glycans are also believed to protect this glycocin fromproteolysis. The plantaricin ASM1 of L. plantarum A-1 and lan-tibiotic sublancin 168 of B. subtilis 168 are also believed to be(S-)glycosylated (272, 273).

S-layer proteins. S-layer proteins form the outermost cell en-velope of some bacterial species. These crystalline layers are madeup of identical protein or glycoprotein subunits. The lattice-like

S-layers are formed by an entropy-driven self-assembly process oftheir substituent (glyco)proteins. The mono- or multilayered S-layers confer an evolutionary advantage to the cells but are notessential for their survival (539). Their self-assembling capacitiesmake them interesting candidates for study in the field of biomi-metics and glycoengineering of therapeutic targets (e.g., vaccinedesign, drug targeting, and diagnostics) (540). The research fielddedicated to these intriguing molecules is called “nanoglycobiol-ogy” (541).

It was long believed that only Gram-positive species could pro-duce S-layers, but the first Gram-negative S-layer was discoveredin 2006 in Tannerella forsythia (502). S-layers are common inBacillaceae such as Clostridium species and Bacillus species, withBacillus stearothermophilus being a model organism (541). Manylactobacilli also produce this crystalline layer (542).

S-layers have been excellently reviewed (539, 541, 543), and weprovide only a short overview of the current knowledge on theirstructure and biosynthesis mechanisms in relation to their glyco-sylation.

Glycans on S-layer glycoproteins can be linear or branchedlong homo- or heteropolysaccharides composed of identical re-peating units. In the bacteria studied to date, these oligosaccha-rides are O-linked to a serine or threonine, although linkages totyrosine residues are also found. Commonly, one glycan is foundper protein, which accounts for 1 to 10% of the molecular weightof the S-layer proteins. Fifty to 150 sugar monomers (hexoses,amino sugars, and exotic sugars, etc.) can be found in 15 to 50repeating units of 2 to 6 sugars.

S-layer glycans resemble O-antigen-carrying LPS molecules, asthey consist of a tripartite structure in which the glycan chain of avariable number of repeating units (cf. O antigen) is linked via avariable core oligosaccharide to the S-layer protein backbone(versus lipid A in LPS). The oligosaccharide is strain specific, whilethe core is less variable. In some cases, the core oligosaccharide ismissing, and the sugar is attached directly to the protein. Thelinkage sugar, i.e., the first repeating unit, can have an invertedanomeric configuration, in which case it is called a pseudocore. Anoncarbohydrate constituent, such as an O-methyl group, can capthe terminal sugar of the oligosaccharide. This constituent poten-tially serves as a termination signal, analogous to LPS biosynthesis(see above). The proteins are water insoluble and have multipledomains: the cell wall-targeting region (anchoring in the PG) andthe self-assembly domain (541).

Nucleotide-activated sugars are used in the biosynthesis of S-layer glycoproteins, and assembly of the oligosaccharide requires aUndP carrier. Genes involved in S-layer glycosylation are in somespecies clustered in an “S-layer glycosylation cluster” (slg), as ded-icated systems. This cluster harbors a priming GT, a transporter, apolymerase, and an OST, thus being analogous to en bloc N-gly-cosylation. Some clusters contain a flippase, which would pointtoward a mechanism resembling Wzy-dependent O-antigen bio-synthesis.

COMMONALITIES IN GLYCOCONJUGATE BIOSYNTHESIS

The plethora of glycoconjugates produced by bacteria is synthe-sized by only a limited number of biosynthesis pathways, as elab-orated extensively above. These pathways share important generalthemes, such as homologous proteins, which point toward an evo-lutionary connectivity in glycoconjugate biosynthesis strategies.Several organisms even modify several of their glycoconjugates



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with the same glycans or have enzymes that are active in more thanone biosynthesis pathway.

General Themes in Glycoconjugate Biosynthesis Pathways

The two most obvious general themes in glycoconjugate biosyn-thesis pathways have been conceptualized as described above: thesequential (Fig. 1) and en bloc (Fig. 2) pathways. Both pathwaysare extensively illustrated by using examples of several bacterialglycoconjugates (Fig. 4 to 8). Here, we zoom in on the commondenominators of both biosynthesis pathways.

The lipid carrier. The biosynthesis of almost all glycoconju-gates requires the involvement of a lipid carrier on which the gly-can moiety is built prior to its transport to its substrate. In mostcases, this lipid carrier is a UndP carrier: O antigen of LPS, CPS,EPS, TA, PG, and en bloc protein glycosylation (46). Even Myco-bacterium species that have a modified PG layer (arabinogalactan)use a modified version of the UndP carrier for the assembly oftheir PG, decaprenol phosphate (544). However, some sequentialglycoconjugate biosynthesis pathways use membrane lipids suchas diacylglycerol and phosphatidylinositol as a carrier (e.g., CPSbiosynthesis in E. coli K1) (69), and in the case of some mem-brane-spanning synthases, no lipid carrier is needed (e.g., alginatebiosynthesis) (40, 71).

A priming GT transfers the first sugar moiety to the lipid car-rier and catalyzes the formation of a high-energy pyrophosphatebond. The resulting undecaprenyl pyrophosphate (UndPP) lipidcarrier serves as a scaffold for the further synthesis of the glycan.The lipid-linked oligosaccharide is then transported over themembrane, the phosphate-sugar bond is cleaved, and the oligo-saccharide is transferred to its substrate. The lipid carrier is thenrecycled to the cytoplasmic face of the membrane (46). A phos-phatase (e.g., Wzb) removes the remaining extra phosphate afterthe transfer of the glycan (76, 111). The recycling of the UndP lipidcarrier is essential for the viability of bacterial cells because of itskey role in PG biosynthesis (39).

As stated above, the factors determining the substrate (accep-tor) and donor specificity of GTs remain largely unclear, but it isbelieved that priming GTs and OSTs recognize the lipid carriers(e.g., length and saturation, etc.) (13). An important exception isthe PglL OST of Neisseria, which is known for its extreme promis-cuity in view of the lipid-linked oligosaccharides used (66).

Priming or initiating GTs. The activated lipid-linked sugarmonomer resulting from the action of the priming GT forms theacceptor for the action of other GTs that assemble the glycan fur-ther (47).

As illustrated in the discussion of the biosynthesis of LPS struc-tures (see above), there are currently two families of priming GTscharacterized. The first family is the polyprenyl-P N-acetylhex-osamine-1-P transferase (PNPT) family, of which the E. coli WecAenzyme is a type example (47, 48, 75, 108) (Fig. 6B). The secondfamily is the family of polyprenyl-P hexose-1-P transferases(PHPTs), and its best-studied member is the WbaP priming GT ofS. enterica serovar Typhimurium (49, 545) (Fig. 6A). These fami-lies are evolutionarily unrelated and have different topologies andprimary sequences. The PNPT family, besides LPS O-antigen bio-synthesis, is often implicated in TA (e.g., TagO of B. subtilis [546]),PG (e.g., MraY [547]), and glycoprotein (e.g., WbpL of P. aerugi-nosa [175]) biosynthesis. The PHPT family can initiate O-antigenLPS (e.g., S. enterica) and EPS/CPS (e.g., various E. coli strains)

biosynthesis but also glycoprotein biosynthesis (e.g., Neisseriaspecies) (478).

Transport of the lipid-linked oligosaccharide across themembrane. There are two main strategies used to transfer lipid-linked glycans across the membrane: flippases and ABC transport-ers. Both are discussed above in detail for EPS/CPS and LPS O-an-tigen biosynthesis (Fig. 5 and 6) but are also involved in thebiosynthesis of other glycoconjugates, such as PG (Fig. 3), TAs(Fig. 7), and glycoproteins (Fig. 8A and D).

The Wzx flippase system generally transfers short, complex,and often branched oligosaccharides and exhibits low substratespecificity (548). ABC transporters are involved in the transloca-tion of longer glycan chains (549). The first system is believed torecognize the first sugar linked to the lipid carrier (550), whileABC transporters recognize the stop elongation signal, such asglycan caps or terminal methylation (549, 551). The ABC trans-porters can consist of a single polypeptide chain (e.g., PglK of C.jejuni) or several polypeptide chains (e.g., Wzt and Wzm of E. coli)(321).

Glycan ligation to the substrate. In the end, the glycan has to beconjugated to its substrate, being lipid A-core polysaccharide (Oantigen), polysaccharide (EPS/CPS and O antigen), or protein(glycoprotein). Strikingly, these reactions are often catalyzed byevolutionarily connected enzymes, all harboring a Wzy_C domain(51). These enzymes include WaaL O-antigen ligases and O-OSTsinvolved in protein glycosylation. N-OSTs, like the PglB N-OST ofC. jejuni, seem to be unrelated and of different evolutionary ori-gins (see above) (13). These enzymes remove the glycan from itslipid carrier by cleaving the phosphate-sugar bond and form a newlinkage with the substrate (51, 150, 392). Nevertheless, it is cur-rently very difficult to predict the substrate specificity, i.e., theglycoconjugate biosynthesis pathways in which these enzymes areinvolved, merely from sequence information. This often results inmisannotations in full genome sequences (e.g., the annotation ofGTs implicated in LPS biosynthesis in Gram-positive species) and,thus, probably also in metagenomic analyses.

Common themes facilitate new discoveries. The fact that al-most all biosynthesis pathways use homologous carriers, enzymes,and transporters reflects the importance of the maintenance ofthese pathways from an evolutionary perspective. There is also anevolutionary connection to the eukaryotic glycobiosynthesispathways, but an in-depth comparison of prokaryotic and eukary-otic glycosylation is beyond the scope of this work, and this is thetopic of a previous review (279).

There are still many species for which the glycosylation poten-tial has to be unraveled. New emerging techniques in MS (309,311, 552), nuclear magnetic resonance (NMR) (327), analyticalglycoscience (553), single-molecule force spectroscopy (554),shotgun glycomics (555), and in silico glycobiology (269) boostthe further in-depth study of new and already known glycoconju-gates (130). The already known common themes can facilitatethese studies and also make it easier to spot important exceptionsand evidence that is still lacking. An example is found in B. fragilis,where a Wzx transporter, but so far, to the best of our knowledge,no OST, has been identified (270).

Overlap in Glycoconjugate Biosynthesis Pathways

In several species, the commonalities between the biosynthesispathways of their glycoconjugates are even more elaborated. Sev-eral species use the same oligosaccharides to build up their LPS

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and glycoprotein structures. Other species harbor pathways en-compassing shared enzymes. The most common overlapping gly-coconjugate biosynthesis mechanisms are the polysaccharide(CPS, LPS, and LOS) and glycoprotein biosynthesis pathways.There are two main trends, the first one being the attachment ofthe same glycan moieties to both glycoconjugates and the otherbeing competition for sugar precursors.

The first phenomenon, i.e., double usage of the same glycanand, thus, the same biosynthesis pathway to modify different gly-coconjugates, has already been illustrated for several species. A.baumannii has only one priming GT, PglC, to initiate CPS andO-glycoprotein biosynthesis (518). This results in the attachmentof pentasaccharide CPS building blocks to glycoproteins (maxi-mum of two linked subunits). A single locus is thus responsible forthe production of both glycoconjugates (518, 556). In C. jejuniNCTC 11168, both CPS and LOS consist of heptoses, which re-quire the activity of the same GmhB phosphatase (114, 557). Thesame glycan is also incorporated into the CPS and LPS glycans ofE. coli O9a:K30 (61). However, most examples of shared glycansare found between overlapping LPS and glycoprotein biosynthesispathways. In P. aeruginosa 1244, the pili are glycosylated with thesame building blocks as those used for LPS O-antigen biosynthesis(386, 387). Both pathways are distinguished later by the differencein specificities and active sites of the competing enzymes Wzy,WaaL, and PilO (390). The EmaA adhesin of A. actinomycetem-comitans is modified with O-antigen sugars and is a substrate ofthe WaaL enzyme (437–439). The same is true for B. pseudomallei,where the glycan attached to the RgpA glycoprotein is similar tothe anionic polysaccharides (428, 431). In E. coli 2787, the Aah GTuses heptose residues from the LPS core to glycosylate the AIDA-Iadhesin (286).

As mentioned above, a second trend is the competition for nu-cleotide-activated sugar precursors between different glycoconju-gate biosynthesis pathways. In P. aeruginosa strain PAO1, both theLPS and glycoprotein biosynthesis pathways use the TDP-L-Rhaprecursor (558). This is also the case for C. jejuni NCTC 11168,where the same Gne epimerase (CJ1131c) is involved in the pro-duction of Gal and/or GalNAc precursors, which are present on itsN-glycoproteins and LOS and CPS structures (14, 114). Glycosy-lation of the HMW1 adhesin of H. influenzae requires UDP-glu-cose and, thus, the phosphoglucomutase PgmB, an enzyme alsoimportant for LOS biosynthesis (73). The same precursor is usedin B. pseudomallei for LPS biosynthesis and flagellin glycosylation.The RmlB enzyme of the LPS O-antigen cluster synthesizes thisnucleotide-activated sugar precursor (537).

These overlapping enzymes and pathways are particularly in-triguing, as they provide the bacteria with several advantages butalso disadvantages. As mentioned above, the production of glyco-conjugates is an energy-costly process for bacteria. Commonali-ties in glycosylation pathways reduce the arsenal of precursors andenzymes needed and are thus energy saving. Bacteria also need away to discriminate between these bifurcated biosynthesis path-ways and have evolved several solutions to do so: they can expressonly a subset of the glycoconjugate repertoire (e.g., H. pylori[322]), use downstream enzymes with different specificities (e.g.,P. aeruginosa [393]), and have both pathways compete (e.g., thesame precursors for LOS, CPS, and N-glycoprotein biosynthesisin C. jejuni [14]), and their expression can be dependent on envi-ronmental stimuli (e.g., expression of colonic acid in E. coli [65]).

Common glycans can serve as a cloak to shield immunogenic

molecules from the host immune system (e.g., P. gingivalis [430]).Some species exploit this by covering their surface with glycansresembling host sugars (e.g., fucose and sialic acid, etc.). Thisstrategy is used by commensals such as B. fragilis, which harbors asurface fucosylation pathway to modify its CPS and O-glycopro-teins (139), but pathogens such as H. pylori also use this strategy toprolong their presence in the host (322). However, the addition ofsimilar antigenic glycans to more structures can also have a majordisadvantage: it can enhance recognition by the immune system.In P. aeruginosa 1244, for instance, O-antigen subunits are alsoadded to the pilin, enlarging the pool of antigens and activatingthe cross-reaction of antibodies (387).

Bifunctional and Promiscuous GTs

Another common theme in glycoconjugate biosynthesis is the in-volvement of bifunctional and promiscuous GTs in several bacte-ria. Bifunctional GTs combine the catalysis of the formation of aglycosidic linkage with another catalytic role. Classic examples arethe bifunctional PBPs used in PG biosynthesis, which have both atransglycosylase and a transpeptidase domain (39). In L. monocy-togenes, GmaR (Lmo0688) is a bifunctional GT, which also tran-scriptionally regulates flagellar expression (see above) (379). ThePglB priming GT of N. gonorrhoeae and N. meningitidis is both anacetyltransferase and a phosphoglycosyltransferase (478). How-ever, the unrefuted champions of multitasking are of course thesynthases, which initiate, elongate, and transport glycans (41).

Promiscuous enzymes can transfer a plethora of glycans and/orcan target several substrates. Most promiscuous enzymes areOSTs, with PglL of N. meningitidis MC58 being an exponent ofthis, as this OST can transfer virtually any glycan (66, 67). Theenzyme can even interfere with the PG machinery and transferincomplete PG subunits (muramoyl-GlcNAc tetra- and tripep-tides) to pilin (66). PglL can even act as a Leloir enzyme andtransfer monosaccharides (68). The same phenomenon could alsobe linked to the PglB N-OST of C. jejuni, the only requirement ofwhich is the presence of a C-2 acetamido group in the nonreduc-ing sugar (53, 323). The PilO O-OST is also promiscuous but to alesser extent (see above) (392). An example of the promiscuity ofa non-GT is the fact that the PglK transporter of C. jejuni cancomplement a Wzx defect (321). Moreover, a homologue of PglK(Wzk) was identified as the O-antigen ligase in H. pylori (322).

CONCLUSIONS

The field of bacterial glycosylation remains enigmatic due to thecomplexity of the field. The current knowledge summarized hereprobably only scratches the surface of what there is to know aboutbacterial glycobiology. Bacterial glycoconjugates exhibit an enor-mous diversity, but their biosynthesis is also based on commonthemes and pathways. This common ground can enhance thestudy of the still unexplored glycosylation potential of several bac-terial strains but should be complemented with efficient analyticalmethods, since sequence information does not adequately predictthe glycosylation potential.

Although glycoconjugates such as EPS, CPS, and LPS havebeen studied for years, many aspects remain unknown. In addi-tion, research concerning the functional importance of glycocon-jugates such as glycoproteins has especially been lagging behind.Currently, the focus lies mainly on the elucidation of their role invirulence, pathogenesis, and bacterium-host interactions in gen-eral. However, their functional importance can be even more vi-



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brant than currently appreciated. For instance, several groups re-ported the (potential) glycosylation of proteins involved in themultienzyme complexes of the cell wall biosynthesis and divisionmachinery (270, 510, 569). It is tempting to speculate that theas-yet-unexplored glycosylation of major enzymes in these com-plexes could play a role in the regulation of their interaction andactivity, but this remains to be elucidated. The few illustrations ofthe fascinating functional importance of bacterial protein glyco-sylation are the modulation of flagellin glycosylation in H. pylorito remain stealth from the immune system (363); the link betweenprotein glycosylation, redox reactions, and electron transport sys-tems seen in Neisseria species (471, 488), and the dynamic inter-play between posttranslational modifications in N. gonorrhoeae(484).

Another important driver of the further expansion of the fieldof prokaryotic glycobiology is its glycoengineering potential. Thistechnology is extremely interesting for industrial applicationssuch as the amelioration of food quality by the engineering of EPSstructures or the production of engineered glycoproteins, such asvaccines and human therapeutic agents (e.g., insulin) (131, 559–568). A better knowledge of the glycosylation mechanisms in ben-eficial bacteria could cause a revolution in the biosynthesis of ther-apeutic molecules in prokaryotic vectors.

The current knowledge on bacterial glycosylation probablyonly scratches the surface of what there is to know. However, thealready known facts show that further elucidation of the enigmaticworld of bacterial glycoconjugates is worth pursuing, as this canrender important insights into bacterium-host interactions andour understanding of bacterial life. Without a doubt, the future ofbacterial glycobiology looks promising and exciting.

ACKNOWLEDGMENTS

We gratefully acknowledge J. Vanderleyden for infinitely supporting ourresearch on Lactobacillus cell wall biology. H.L.P.T. acknowledges PieterVanbosseghem for help with the figures.

H.L.P.T. also acknowledges the Institute for the Promotion of Inno-vation through Science and Technology in Flanders (IWT Vlaanderen)for her Ph.D. scholarship. S.L. previously held a postdoctoral grant fromthe Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO) to studyprotein glycosylation in lactobacilli. We are also grateful for the financialsupport of the University of Leuven via grant PF3M100234, FWO Vlaan-deren (Krediet aan Navorsers 28960 to S.L.), and UAntwerpen (BOF).

We declare no conflicts of interest and apologize to the authors whosework was not included in this work.

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