Unique lipid anchor attaches Vi antigen capsule to thesurface of Salmonella enterica serovar TyphiSean D. Listona, Olga G. Ovchinnikovaa, and Chris Whitfielda,1

aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G2W1

Edited by Hiroshi Nikaido, University of California, Berkeley, CA, and approved April 29, 2016 (received for review December 14, 2015)

Polysaccharide capsules are surface structures that are critical forthe virulence of many Gram-negative pathogenic bacteria. Salmo-nella enterica serovar Typhi is the etiological agent of typhoidfever. It produces a capsular polysaccharide known as “Vi anti-gen,” which is composed of nonstoichiometrically O-acetylatedα-1,4-linked N-acetylgalactosaminuronic acid residues. This glycanis a component of currently available vaccines. The genetic locusfor Vi antigen production is also present in soil bacteria belongingto the genus Achromobacter. Vi antigen assembly follows a wide-spread general strategy with a characteristic glycan export stepinvolving an ATP-binding cassette transporter. However, Vi anti-gen producers lack the enzymes that build the conserved terminalglycolipid characterizing other capsules using this method. Achro-mobacter species possess a Vi antigen-specific depolymerase en-zyme missing in S. enterica Typhi, and we exploited this enzyme toisolate acylated Vi antigen termini. Mass spectrometry analysisrevealed a reducing terminal N-acetylhexosamine residue modifiedwith two β-hydroxyl acyl chains. This terminal structure resemblesone half of lipid A, the hydrophobic portion of bacterial lipopolysac-charides. The VexE protein encoded in the Vi antigen biosynthesislocus shares similarity with LpxL, an acyltransferase from lipid A bio-synthesis. In the absence of VexE, Vi antigen is produced, but itsphysical properties are altered, its export is impaired, and a Vi capsulestructure is not assembled on the cell surface. The structure of thelipidated terminus dictates a unique assembly mechanism and haspotential implications in pathogenesis and vaccine production.

polysaccharide capsule | Vi antigen | Salmonella | glycolipid |polysaccharide export

Many bacteria produce high-molecular-weight cell-surfacepolysaccharides that form a hydrated layer known as a

“capsule.” There is enormous diversity in capsular polysaccha-ride (CPS) structures resulting from variations in sugar residuecomposition, linkage(s), and the addition of nonsugar substituents(1). The capsule is often the outermost structure of a bacterial celland therefore is critical for interactions with the environment.Depending on the organism, capsules assist bacteria in resistingdesiccation, forming biofilms, colonizing host tissues, resistingbacteriophages, and reducing opsonophagocytosis and comple-ment-mediated killing (2). In Salmonella enterica, the virulencecapsular polysaccharide, known as “Vi antigen,” is produced byhuman-restricted serovar Typhi (hereafter S. Typhi, the etio-logical agent of typhoid fever) and serovar Paratyphi C, but it isabsent in other serovars commonly associated with gastroenteritis.Vi antigen capsule is implicated in the evasion of the innate im-mune system (reviewed in ref. 3). The production of Vi antigenreduces serum complement binding/killing and promotes intracel-lular replication; Vi antigen-deficient mutants are 10,000-fold lessvirulent in a mouse model of infection (4). Purified Vi antigen iscurrently used in parenteral vaccines (5).Despite the structural diversity of Gram-negative CPS, most

are synthesized by one of two widely distributed mechanisms,with model systems provided by Escherichia coli K antigens(reviewed in ref. 1). The two pathways are differentiated by themechanism and location of the polymerization reaction and bythe machinery that exports the nascent glycan (or its biosynthetic

intermediates) across the cytoplasmic membrane. One of thesesystems requires a pathway-defining ATP-binding cassette (ABC)transporter to export CPS that is fully polymerized in the cytoplasm.This mechanism is shared by extraintestinal pathogenic E. coli (i.e.,group 2 CPS), Neisseria meningitidis, Haemophilus influenzae,Campylobacter jejuni, and other pathogens of humans and livestock.In these bacteria, the CPS glycans are attached to a (lyso)phosha-tidylglycerol moiety via an oligosaccharide of five to nine β-linked3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues (6). The Kdo-containing glycolipid is synthesized by conserved β-Kdo transferases(known as “KpsS” and “KpsC” in E. coli) (7). The CPS glycan isbuilt on the nonreducing end of the β-Kdo oligosaccharide linker byserotype-specific glycosyltransferases. The lipidated terminus isessential for CPS export (6) and is likely recognized by thepathway-defining ABC transporters, which are interchangeableamong CPS serotypes and species (reviewed in refs. 1 and 8).CPS translocation to the cell surface is believed to involve anenvelope-spanning complex composed of the ABC transporterand members of the polysaccharide co-polymerase (PCP) and outermembrane polysaccharide export (OPX) protein families (1, 8).Vi antigen is a linear polymer of GalNAcA residues non-

stoichiometrically O-acetylated at C-3 (9). The Vi antigen bio-synthesis (viaB) operon encodes enzymes implicated in Vi antigenbiosynthesis (TviA–E) as well as a characteristic ABC transporter(VexBC) and OPX (VexA) and PCP (VexD) homologs (Fig. 1A)(10, 11). Loci similar to S. Typhi viaB are found in the opportu-nistic pathogens Citrobacter freundii (12), Bordetella petrii (GenBankaccession no. AM902716.1), and Achromobacter species (Fig.1A), although the glycan product has not been investigated ineither Bordetella or Achromobacter. The chromosomes of the Viantigen-producing bacteria lack homologs of kpsS or kpsC that arefound in all other currently known Gram-negative bacteria withABC transporter-dependent CPS assembly pathways (8). Here we

Significance

Polysaccharide capsules are protective surface layers that en-hance virulence of many pathogenic bacteria. Salmonella entericaserovar Typhi is the causative agent of typhoid fever, and itproduces the virulence capsular polysaccharide known as “Viantigen.” This glycan is part of some current vaccines. In someGram-negative bacteria, capsular polysaccharides are attached toa conserved glycolipid that anchors the polysaccharide to the cellsurface and is required for its transport across the cell envelope.S. enterica Typhi follows a different strategy; this work identifiesa reducing terminal lipid structure unique to the Vi antigen that isrequired for attachment of the capsular surface layer. This lipid isstructurally (and potentially biosynthetically) related to the con-served lipid A component of bacterial lipopolysaccharides.

Author contributions: S.D.L., O.G.O., and C.W. designed research; S.D.L. performed research;S.D.L., O.G.O., and C.W. analyzed data; and S.D.L. and C.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence reported in this paper has been deposited in the GenBankdatabase (accession no. KT99772).1To whom correspondence should be addressed. Email: cwhitfie@uoguelph.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1524665113/-/DCSupplemental.

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identify the structure of a glycolipid terminus unique to the Viantigen and propose a biosynthetic origin that takes advantage ofthe conserved lipid A machinery in Gram-negative bacteria.

ResultsVi Antigen from a ΔvexE Mutant Has Altered Physical Properties.Despite the absence of kpsS and kpsC from the chromosomesof viaB-positive bacteria, we speculated that a glycolipid terminusof some form may be a unifying feature for all ABC transporter-dependent CPS biosynthesis pathways. Other bacterial surfaceglycoconjugates exported by ABC transporters frequently useundecaprenyl diphosphate carrier lipids. To test the possibilitythat these lipids participated in Vi antigen production, the viaBlocus was introduced into E. coli CWG1214 ΔwecA, which isunable to make undecaprenyl diphospho-N-acetylglucosamine inthe obligatory first step in biosynthesis of E. coli LPS O antigensand enterobacterial common antigen (13). E. coli also lacks wbaP,whose gene product produces undecaprenyl diphospho-galactosein the corresponding initiation step for most Salmonella O antigens(13). E. coli CWG1214, transformed with the viaB locus, displayedrobust Vi antigen production, evident in immunoblots (Fig. 1E),ruling out the logical candidates for undecaprenyl-active enzymesin Vi antigen assembly. All the viaB loci encode a predicted VexEprotein containing a potential C-terminal lysophospholipid acyl-transferase (LPLAT) motif (Fig. 1B). This motif is also found in E.coli LpxL (Fig. 1C), an acyl carrier protein (ACP)-dependent sec-ondary acyltransferase involved in the biosynthesis of LPS lipid A,a conserved glycolipid essential for viability of almost all Gram-negative bacteria (reviewed in refs. 13 and 14) (see Fig. 4). LpxLand VexE share only 18% identity overall (e-value: 1.08 × 10−3), butthe conserved LPLAT domain (cd07984) shares higher similarity

(e-value: 1.67 × 10−8). The putative LPLAT motif in VexE led tothe hypothesis that this protein is an acyltransferase that creates adifferent type of lipid terminus on Vi antigen chains.Previous analyses of Vi antigen phenotypes in viaB gene mu-

tants were performed using recombinant E. coli transformed withplasmid-encoded viaB, but the possibility of a lipid terminus andthe precise role of VexE has not been examined (10, 11). Toavoid complications arising from multicopy gene expression andthe unnatural host background, we examined the role of VexEusing chromosomal mutations in S. Typhi. In Western immu-noblots, Vi antigen in cell lysates of the parent strain bound toboth hydrophobic PVDF and positively charged nylon mem-branes (Fig. 1D). In contrast, Vi antigen in lysates from theΔvexE mutant bound only to nylon, indicating a change in thephysical properties of Vi antigen produced by the mutant. Wild-type binding properties were restored in the mutant by the ex-pression of VexE homologs from S. Typhi or Achromobacterdenitrificans (Fig. 1D). LPLAT enzymes possess a conservedHX4D/E motif, which contains the essential catalytic His/Asppair (Fig. 1C) (15). The corresponding H→A mutant in E. coliLpxL results in a >1,000-fold reduction in lauroyltransferaseactivity (16). The putative catalytic His residue was mutated inthe VexE homologs from S. Typhi and A. denitrificans, and theseproteins were expressed in S. Typhi ΔvexE. Vi antigen from thesetransformants did not bind to PVDF despite robust expression ofthe enzymes (Fig. 1D). Proper folding of the mutant VexE wasconfirmed by comparing circular dichroism spectra of purifiedwild-type and mutant proteins (Fig. S1). The catalytic activity ofVexE therefore is linked to alterations in the physical propertiesof Vi antigens, resulting in differential binding to membraneswith varying chemistries.

A

B

C

D E

Fig. 1. S. Typhi wild-type and ΔvexE mutant Vi antigens possess altered physical properties. (A) Shared organization and gene content in viaB loci. The viaBloci from Achromobacter sp. include an additional gene (vexL) encoding a Vi antigen lyase enzyme. The A. denitrificans sequence is deposited at GenBank(accession no. KT997721), but the same locus is found in other sequenced genomes of Achromobacter sp. [A. xylosoxidans (GenBank accession no.CP012046.1), A. arsenitoxydans (GenBank accession no. NZ_AGUF01000055.1), A. spanius (GenBank accession no. NZ_LGVG01000001.1), and A. piechaudii(GenBank accession no. ADMS01000020.1)]. (B) VexE contains a predicted N-terminal region of tetratricopeptide repeats (TPR), which form α-helical super-structures implicated in protein–protein interactions (25), and a C-terminal lysophospholipid acyltransferase (LPLAT) domain. (C) Multiple sequence alignmentof the LPLAT domain of VexE from S. Typhi, A. denitrificans, and E. coli LpxL. Motifs characteristic of LPLAT are highlighted in yellow, and the putative role ofparticular residues is noted (17). Residues that were mutated are boxed in blue. (D) In immunoblots, Vi antigen produced by S. Typhi bound PVDF andpositively charged nylon membranes, whereas Vi antigen from the ΔvexE mutant bound only nylon. The panels show immunoblots of proteinase K-digestedwhole-cell lysates probed with anti-Vi antigen antibody. PVDF binding was restored when the ΔvexE mutant was complemented with either S. Typhi vexE orA. denitrificans vexE. The corresponding putative catalytic mutants of VexE from either S. Typhi (H487A) or A. denitrificans (H466A) failed to restore PVDFbinding. A Y471F mutation in the A. denitrificans enzyme (at position 6 of HX4(D/E) motif in VexE) had no discernible effect on its activity. VexE expressionwas monitored by Western blotting of hexahistidine-tagged VexE constructs from identical cell cultures. (E) Vi antigen is produced in E. coli Top10 and itsΔwecA mutant.

6720 | www.pnas.org/cgi/doi/10.1073/pnas.1524665113 Liston et al.

To determine whether the altered binding properties reflecteddifferences in the repeat-unit structure of the parental and mu-tant Vi antigens, such as alterations in O-acetylation, CPS waspurified, and its structure was examined by NMR. In the initialpreparations we were unable to obtain wild-type Vi antigen freefrom LPS contamination, and substantial amounts of wild-typeVi antigen sedimented with LPS micelles in centrifugation. De-letion of vexE abrogated this property (Fig. S2B), adding weightto the contention that VexE influenced the physical properties ofVi antigen. To obtain Vi antigen free of LPS, we created ΔwaaGmutants generating truncated LPS molecules (resulting from theloss of most of the core oligosaccharide and O antigen; reviewedin ref. 15) that could be separated from Vi antigen by gel fil-tration chromatography (Fig. S2 A–C). 13C NMR spectra of Viantigens from ΔwaaG and ΔwaaG ΔvexE double mutants wereidentical (Fig. S2 D and E and Table S1) and were comparable tothose previously published (17). The altered properties of the Viantigen produced by the vexE mutant therefore were not causedby changes in the polysaccharide backbone structure but couldbe explained by alterations in a putative acylated terminus.

Vi Antigen Has a Unique Glycolipid at Its Reducing Terminus. Struc-tural investigation of the termini of high-molecular-weight polysac-charides requires a method that reduces the degree of polymeri-zation while preserving linkages between terminal modification(s)and the remaining glycan. Previously, we exploited endoglycanaseenzymes from capsule-specific bacteriophages to identify the ter-minal glycolipid structure from the CPS of E. coli K1 and K5 andmeningococcal serotype b (6). The viaB locus of Achromobacter sp.and B. petrii contain an additional ORF located downstream of vexEin the otherwise similar locus (Fig. 1A). The predicted gene product

shared sequence similarity with known pectate lyase enzymes(Fig. S3A); the corresponding gene is renamed “vexL.” Pectate ly-ases degrade acidic polymers, such as pectin, from plant tissues (18),and the α-1,4-linked polygalacturonic acid backbone structure ofpectin superficially resembles Vi antigen. Purified A. denitrificansVexL enzyme depolymerized Vi antigen (Fig. S3B). Hydrophobicproducts released in these reactions were collected by solid-phaseextraction and were analyzed by LC-MS.LC-MS of these molecules revealed a series of species that

differed by 217.059 m/z, representing increments of one Gal-NAcA residue (Fig. 2A and Table S2). Oligosaccharides modi-fied by one or more O-acetyl groups (δ m/z = 42.011) were alsopresent. Lyase enzymes cleave polysaccharides through an elim-inative mechanism and create a characteristic (anhydro) 4-deoxy-α-D-galact-4-enuronosyl residue at the nonreducing end of theoligosaccharide (18). This modification was evident in MS/MSfragmentation products (Fig. 2B). The Vi antigen oligosaccharideswere linked to a reducing terminal N-acetylhexosamine (HexNAc)residue modified with either two β-hydroxymyristate chains or oneβ-hydroxymyristate and one β-hydroxypalmitate chain. Fragmen-tation of the [M-H]− ion at 1333.666 m/z produced products (in-cluding cross-ring cleavages) consistent with a structure comprisingthree GalNAcA residues linked to a single reducing terminalHexNAc possessing β-hydroxymyristate and β-hydroxypalmitatemodifications (Fig. 2B). As confirmation, the isotopic distribution ofthe [M-H]− ion at 1333.666 m/z agreed with that predicted for theglycolipid (Fig. S4A), and fragmentation of the [M-2H]2- ion atm/z =774.858 revealed the same structure extended with an additionalGalNAcA residue (Fig. S4B). No ions corresponding to this gly-colipid were identified when the procedure was repeated for Viantigen purified from a ΔvexE mutant of S. Typhi.

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Fig. 2. Glycolipid terminus of the Vi antigen as determined by MS. (A) Charge deconvoluted LC-electrospray ionization (ESI)-QTOF-MS spectrum in negativemode for Vi antigen termini purified from S. Typhi. All ions correspond to a di-β-hydroxyacylated HexNAc residue linked to two or more variably O-acetylatedHexNAcA residues. (B) LC-ESI-QTOF-MS/MS data for the singly charged (blue) and doubly charged (red) ions corresponding to a GalNAcA3 oligosaccharideattached to a reducing terminal diacyl-HexNAc. Overlapping signals are colored purple. Fragmentations are illustrated in green.

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VexE Is Required for Efficient Export and Cell-Surface Retention of ViAntigen. The role of the glycolipid terminus in Vi antigen exportand surface assembly of the Vi capsule was investigated inS. Typhi by its susceptibility to degradation by VexL. VexL de-graded almost all detectable Vi antigen in the wild type, indicatingminimal amounts of intracellular (untransported) glycan. In con-trast, Vi antigen profiles from the ΔvexC (lacking the ABC trans-porter ATPase) or ΔvexE mutant were unaffected by the presenceof the enzyme (Fig. 3A). Vi antigen stability in whole cells wascaused solely by inaccessibility to the lyase, as permeabilization ofthe mutant cells facilitated complete digestion of the glycan. Fur-thermore, a Vi antigen-specific bacteriophage infected the wild typebut formed no plaques on the ΔvexC or ΔvexE mutants, confirmingthat no phage receptor is available on the mutant cell surfaces (Fig.S5B). Complementation of the mutations with the respective genesrestored phage sensitivity. Wildtype S. Typhi possessed a Vi antigencapsule on the cell surface that was detectable by immunofluores-cence microscopy (Fig. 3B), and complementation of theΔvexC andΔvexE mutants with the respective genes restored the Vi antigencapsule. Both the ΔvexC and ΔvexE mutants possessed inclusionbodies (Fig. 3B, Insets), which were labeled with Vi antigen-specificantibodies in immunofluorescence microscopy of permeabilizedcells. Electron microscopy revealed that the inclusions were cyto-solic in both the ΔvexC and ΔvexE mutants (Fig. S5C). To examinepossible deleterious effects of these inclusions on cell physiology,the Cpx envelope stress response was assessed in S. Typhi andmutant derivatives (Fig. S5D). Surprisingly, the Cpx response wasup-regulated significantly only in the ΔvexC mutant, in which noexport occurs, and this increase was eliminated in a ΔvexC ΔvexEmutant, indicating that activation of a stress response by the in-clusions was dependent on acylation.These results are consistent with the intracellular accumulation of

Vi antigen in the ΔvexC and ΔvexE mutants, suggesting that bothmutations resulted in export defects. However, published mutantphenotypes indicated extensive Vi antigen export in a vexE mutantof an E. coli recombinant containing viaB (11). Because lyasetreatment and immunofluorescence microscopy of whole cellscannot account for Vi antigen released into the growth medium, weexamined the cell-free supernatants from early exponential-phasecultures of S. Typhi and its mutant derivatives for Vi antigen release(Fig. 3C). Wild-type cells released some Vi antigen into the me-dium, as expected with any encapsulated bacterium and as is con-sistent with published observations (10, 11). The ΔvexC mutantreleased only a trace of Vi antigen; release could be explained bysmall amounts of lysis during growth and is consistent with the re-lease of cytosolic RNA polymerase in the same cultures (Fig. 3C).Export and release of Vi antigen was restored when the ΔvexCmutation was complemented with vexC. In contrast, ΔvexE cellsreleased large quantities of Vi antigen. This material was eliminatedin a ΔvexCE double mutant (Fig. 3C and Fig. S5E), indicating anactive process involving the ABC transporter rather than elevatedleakage resulting from the vexE defect.

DiscussionVi antigen has a lipid terminus that differs from those of anyother known CPS assembled in an ABC transporter-dependentpathway. It is composed of a reducing terminal HexNAc residuemodified with two β-hydroxy fatty acids and resembles one halfof the structure of lipid A (Fig. 4). This structure, together withthe similarity shared by VexE and LpxL, a secondary acyl-ACP–dependent acyltransferase from lipid A biosynthesis, is consistentwith the proposal that VexE is an acyltransferase that transfers aβ-hydroxymyristate or β-hydroxypalmitate chain to the terminusof Vi antigen. The action of VexE would be comparable to thatof the secondary acyltransferases in lipid A biosynthesis, althoughLpxL and LpxM transfer nonhydroxylated fatty acids (Fig. 4) (16).VexE is the only acyltransferase encoded by the viaB locus. Thereis no precedent for such enzymes being able to transfer both acylchains, and doing so would require radically different acceptorspecificities in a single catalytic site. A logical origin of this ter-minal moiety involves secondary acylation of the UDP-activated

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Fig. 3. VexE is required for efficient export and surface retention of Vi an-tigen. (A) The Vi antigen depolymerase was unable to access Vi antigen withinintact cells of the ΔvexE mutant. Whole or lysed cells of S. Typhi and mutantswere incubated with purified VexL, collected, digested with proteinase K, andprobed for Vi antigen. VexL was able to degrade the Vi antigen in wild-typeS. Typhi but not in S. Typhi ΔvexC, providing positive and negative controls forexport, respectively. (B) Immunofluorescence microscopy of cells probed withanti-Vi antigen antibodies illustrated that the ΔvexE mutant possessed no Viantigen on its surface but accumulated intracellular Vi antigen in inclusionbodies, which became accessible to antibody in permeabilized cells. (Scalebars, 10 μm.) Insets are enlarged to show a representative cell. (C) S. TyphiΔvexE was able to export Vi antigen in a transporter-dependent manner.Growth medium from early exponential-phase cultures was collected andprobed for Vi antigen and (cytosolic) RNA polymerase by Western immuno-blotting.

6722 | www.pnas.org/cgi/doi/10.1073/pnas.1524665113 Liston et al.

β-hydroxymyristoyl-GlcNAc product of LpxA in lipid A bio-synthesis (Fig. 4) (19). Such a reaction could divert this intermediatefor use in Vi antigen biosynthesis and, to our knowledge, wouldrepresent the first off-pathway use of an Lpx-pathway in-termediate. We investigated the possibility that VexE interferedwith normal lipid A biosynthesis in E. coli. Expression of VexEslowed the growth of E. coli slightly, but this effect was likelycaused by protein overexpression and was independent of VexEcatalytic activity (Fig. S6A). In addition, VexE was unable tomodify lipid A (Fig. S6B). The inability of VexE activity to in-fluence lipid A biosynthesis is perhaps not surprising, given theregulation of the essential Raetz pathway process. The LpxAreaction equilibrium favors the reverse reaction, and the firstcommitted step of lipid A biosynthesis (LpxC) is tightly regulated(15, 16, 20), so pathway flow is regulated according to lipid Arequirement. We pursued the possibility that ΔvexE Vi antigenpossesses a single acyl chain (the product of LpxA) at its reducingterminus. However, we were unable to detect either diacyl- ormonoacyl-HexNAc in either extracellular or intracellular (accu-mulated) Vi antigen from the ΔvexE mutant. This negative resultcould reflect an absolute requirement for diacylated UDP-GlcNAc,offering an additional means of separation from the lipid A path-way. However, we cannot rule out technical issues in which mon-oacylated Vi antigen termini lack sufficient hydrophobicity forseparation protocols (consistent with altered PVDF binding).The apparent ability to synthesize Vi antigen in the absence

the acylated terminus could reflect the mutations creating con-ditions that facilitate polymer synthesis on nonphysiological ac-ceptors, as is the case in the E. coli and N. meningitidis kpsS andkpsC mutants (6). However, this assumption requires that thediacyl-HexNAc actually serves as an acceptor, but the identityand mechanism of the Vi antigen polymerase is unknown. Viantigen potentially could be synthesized by growth at the re-ducing terminus in a process similar to class I hyaluronan syn-thases. These enzymes use UDP-GlcNAc or UDP-glucuronicacid (GlcA) as acceptors and the nascent [3)-GlcNAc-β-(1→4)-GlcA-β-(1→]n-UDP chain as the donor during chain extension(21). It is unknown how the terminal UDP moiety is removed inthe final glycan product. In such a scenario, the addition ofdiacyl-HexNAc could represent the last step in Vi antigen bio-synthesis before export, explaining the ability to synthesize Viantigen in the absence of VexE. Biochemical characterization ofthe Vi antigen polymerase(s) is required to resolve this question.In the E. coli group 2 CPS assembly, export is dependent on

the presence of the glycolipid terminus (6). In contrast, defectiveacylation of the Vi antigen in the ΔvexE mutant does not preventexport, but accumulation of intracellular Vi antigen (which is notseen in the wild type) also occurs. Accumulation of Vi antigen

could reflect altered recognition by the export machinery. Forexample, the LPS ABC transporter MsbA from E. coli is highlyselective for completed (hexaacylated) LPS molecules. Export oftetraacylated precursors occurs only at low levels (reviewed inref. 13). Alternatively, the export defect in the ΔvexE mutantcould reflect alteration of essential interactions that couplesynthesis to export in a multiprotein complex. However, alteredinteractions are unlikely because the phenotype resulting fromthe VexE catalytic-site mutation (which should preserve protein–protein interactions) is indistinguishable from the vexE deletion(Fig. 3C). Interestingly, intracellular Vi antigen in transport-defective mutants showed an increase in the average chain length(Fig. 3C and Fig. S5A), whereas overexpression of VexE caused areduction. Lowering chain length requires VexE catalytic activityrather than a simple structural requirement for the protein, becausethe size reduction was not evident in ΔvexE cells expressing Vex-EH466A. Altered chain lengths can be explained by an elongationphase differing from the normal assembly process occurring withmolecules with a complete glycolipid terminus. There is precedentfor the modulation of glycan chain length by competition betweenexport and extension in other bacterial systems with ABCtransporters (22, 23).The use of a conserved intermediate from the lipid A–

biosynthesis pathway to create the lipid terminus potentially fa-cilitates Vi antigen production in diverse Gram-negative bacteriaby horizontal transfer of the viaB locus with a limited genecomplement. This diversity is evident in the possession of thelocus by Achromobacter, Bordetella, and Citrobacter sp. and ex-pression in E. coli, but why some Vi antigen producers possessthe additional VexL component is unknown. It is also unknownwhether the terminal lipid itself is important in the interaction ofVi antigen with the host immune system. In the context of Viantigen-based vaccines, a production strain lacking vexE mayoffer advantages because it exports Vi antigen with altered mi-cellar properties and a reduced association with LPS.

MethodsStrains and Plasmids. The bacterial strains used in this study are listed in TableS3. The background for the generation of viaB mutants was S. Typhi H251.1(aroC); clean mutations were generated by recombineering using the λ-redsystem (see SI Methods for details). Strains and transformants were grownat 37 °C in lysogeny broth (LB) medium supplemented with 100 μg/mL 2,3-dihydroxybenzoic acid and antibiotics where appropriate. Complementationof mutations was performed using L-arabinose–inducible pBAD-based vec-tors described in Table S3.

Primers. Oligonucleotide primers used to amplify genes from S. Typhi,A. denitrificans, and E. coli genomic DNA were obtained from Sigma-Aldrichand are described in Table S4.

UDP-GlcNAc

Acetate

LpxC

OHHO

NHHOO

OO UDP

Vi antigen Biosynthesis

UDP-diacyl-GlcN

LpxD

3-OH-C14-ACP

LpxA 3-OH-C14-ACP

OHO

OHHO

NHOO

OO UDP

14

OHO

OHHO

NH2O

O

O UDP

14

O

OHHO

NHOO

OO UDPO

HO

O

14

14/16

7 steps

OHHO

NHO

O

OHO

OHO

O

14 14

UDP

UDP-acyl-GlcNUDP-acyl-GlcNAcUDP-diacyl-GlcNAc

O

OO

NHO

O O

NHOHO

OHO

OO

O

OHO

OO

O

1414141414 12

LpxMLpxL

PO4H2

H2O4P

Kdo2–

VexE?

3-OH-C14/16 -ACP

?

Raetz Pathway

Kdo2 A dipil–

Fig. 4. Proposed model for the biosynthesis of the Vi antigen glycolipid terminus. The diacyl-HexNAc residue at the Vi antigen terminus originates from thesecondary acylation of the UDP-activated acyl-GlcNAc product produced by LpxA in the conserved lipid A biosynthesis Raetz pathway (14).

Liston et al. PNAS | June 14, 2016 | vol. 113 | no. 24 | 6723

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BIOLO

GY

Purification of Vi Antigen. Vi antigen was purified from the supernatant of ahot aqueous phenol extract of lyophilized S. Typhi ΔwaaG and mutant de-rivatives (24). Secreted Vi antigen was precipitated from culture super-natants using hexadecyltrimethylammonium bromide (11). The polysaccharidepreparations were digested with DNase, RNase, and Proteinase K and wereseparated from residual LPS by gel filtration chromatography in the presence ofdetergent (see SI Methods for details).

Isolation of the Vi Antigen Glycolipid Terminus. Twenty milligrams of purifiedVi antigen were resuspended at 1 mg/mL in 50 mM sodium bicarbonate,0.1 mM CaCl2 (pH 7.5). Purified VexL-His6 was added to a final concentration of100 μg/mL; the reaction mixture was incubated at 37 °C for 5 h and then wasloaded into a SepPak C18 cartridge. The column was washed with 10 mL ofwater, and bound hydrophobic material was eluted in 70% (vol/vol) acetonitrile.Eluted material was dried by SpeedVac and was resuspended in 100 μL 25%(vol/vol) acetonitrile in water.

MS. LC-MS analyses of the glycolipid terminus were performed on an Agilent1200 high-performance liquid chromatograph interfaced with an Agilentultra-high-definition (UHD) 6530 quadrupole TOF (QTOF) mass spectrometer.A C18 column was used for chromatographic separation. Conditions for LCand MS are described in SI Methods.

Digestion of Cell Surface and Intracellular VI Antigen with VexL Lyase. Cultureswere grown until OD600 = 0.5 was reached. Cells equivalent to one OD600

unit were collected by centrifugation and were resuspended in PBS sup-plemented with 0.1 mM CaCl2, with and without VexL-His6 (100 μg/mL finalconcentration). Cell suspensions were incubated at 37 °C for 1 h and werecollected by centrifugation. The cells were solubilized in SDS/PAGE bufferand were analyzed by Western immunoblotting using mouse monoclonal

antibody P2B1G2/A9, which is specific for Vi antigen (20). To ensure that theundigested Vi antigen resulted only from its inaccessibility, aliquots of cellswere lysed by French press, unbroken cells were removed by centrifugation,and VexL-His6 was added, incubated, and analyzed as above.

Detection of Cell-Free Vi Antigen in Culture Supernatants. LB cultures (50 mL)were grown at 37 °C until an OD600 of 0.5 was reached. Cells then werecollected by centrifugation at 5,000 × g for 15 min. The supernatant wasdialyzed against water for 2 d, using a dialysis membrane with a 3,500molecular weight cutoff (MWCO). The dialyzed supernatant was lyophilized,resuspended in 1 mL of water, and examined by Western immunoblotting.

Immunofluorescence Microscopy. Live and fixed/permeabilized cells wereprobed with Vi antigen-specific monoclonal antibody (P2B1G2/A9) (20) andwere labeled with rhodamine red-conjugated goat anti-mouse IgG. See SIMethods for details.

ACKNOWLEDGMENTS. Plasmids pGVXN158 containing the viaB locus,pNLP15 containing the spy promoter fused to the luxCDABE cassette,and E. coli BKT09 were generous gifts from Dr. Michael Wetter, Dr.Tracy Raivio, and Dr. Russell Bishop, respectively. We thank Prof. AyubQadri for the gift of monoclonal antibodies raised to Vi antigen; Drs.Dyanne Brewer and Armen Charchoglyan for technical assistance withMS; Mrs. Valerie Robertson and Dr. Andy Lo for technical support withNMR spectroscopy; Dr. Michaela Strüder-Kypke and Mr. Robert Harris forfreeze substitution and electron microscopy; Dr. Colin Cooper for sequencingA. denitrificans; and Dr. Iain Mainprize for the generation of a wecAmutant in E. coli. This work was supported by funding from Canadian Insti-tutes of Health Research. C.W. holds a Canada Research Chair, and S.D.L. is therecipient of a Canada Graduate Scholarship from the National Sciences andEngineering Research Council of Canada.

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