lactobacilli
DESCRIPTION
extracellular biology of lactobacilliTRANSCRIPT
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R E V I E W A R T I C L E
The extracellular biologyofthe lactobacilliMichiel Kleerebezem1,2,3,4, Pascal Hols5, Elvis Bernard5, Thomas Rolain5, Miaomiao Zhou6, Roland J.Siezen1,2,4,6 & Peter A. Bron1,2
1TI Food and Nutrition, Wageningen, The Netherlands; 2NIZO food research, Ede, The Netherlands; 3Laboratory of Microbiology, Wageningen
University, Wageningen, The Netherlands; 4Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands; 5Unite de Genetique,
Institut des Sciences de la Vie, Universite catholique de Louvain, Louvain-la-Neuve, Belgium; and 6Centre for Molecular and Biomolecular Informatics,
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Correspondence: Michiel Kleerebezem,
NIZO food research, PO Box 20, 6710 BA Ede,
The Netherlands. Tel.: 131 0 318 659629;
fax: 131 0 318 650400; e-mail:
Received 8 October 2009; revised 14 December
2009; accepted 14 December 2009.
Final version published online 19 January 2010.
DOI:10.1111/j.1574-6976.2009.00208.x
Editor: Keith Chater
Keywords
Lactobacillus; genomics; exoproteome; cell
wall; host–microorganism interactions;
probiotics.
Abstract
Lactobacilli belong to the lactic acid bacteria, which play a key role in industrial
and artisan food raw-material fermentation, including a large variety of fermented
dairy products. Next to their role in fermentation processes, specific strains of
Lactobacillus are currently marketed as health-promoting cultures or probiotics.
The last decade has witnessed the completion of a large number of Lactobacillus
genome sequences, including the genome sequences of some of the probiotic
species and strains. This development opens avenues to unravel the Lactobacillus-
associated health-promoting activity at the molecular level. It is generally
considered likely that an important part of the Lactobacillus effector molecules
that participate in the proposed health-promoting interactions with the host
(intestinal) system resides in the bacterial cell envelope. For this reason, it is
important to accurately predict the Lactobacillus exoproteomes. Extensive annota-
tion of these exoproteomes, combined with comparative analysis of species- or
strain-specific exoproteomes, may identify candidate effector molecules, which
may support specific effects on host physiology associated with particular
Lactobacillus strains. Candidate health-promoting effector molecules of lactobacilli
can then be validated via mutant approaches, which will allow for improved strain
selection procedures, improved product quality control criteria and molecular
science-based health claims.
Introduction: the lactobacilli
Lactobacilli belong to the lactic acid bacteria (LAB), which
are Gram-positive organisms with a low G1C content that
belong to the phylum of the Firmicutes, and are members of
the Clostridium-Bacillus subdivision of Gram-positive eu-
bacteria (Pot et al., 1994). The genus Lactobacillus currently
includes 148 recognized species (NCBI taxonomy database),
and encompasses an unusually high phylogenetic and func-
tional diversity. Lactobacilli encompass aero-tolerant and
anaerobic species and strains and are classically regarded as
strictly fermentative. They have traditionally been divided
into three groups based on their fermentation characteris-
tics: obligately homofermentative, facultatively hetero-
fermentative and obligately heterofermentative (Pot et al.,
1994; Hammes & Vogel, 1995; Claesson et al., 2008).
However, the presence of heme and/or menaquinone can
stimulate aerobic respiration, leading to increased biomass
formation without acidification in a subset of Lactobacillus
species (Brooijmans et al., 2009).
Many lactobacilli are associated with food and feed
fermentation, mainly because they contribute to raw-mate-
rial preservation due to acidification, but also because of
their capacity to contribute to product characteristics such
as flavor and texture. The natural habitat of lactobacilli
ranges from dairy, meat and plant material fermentations to
the oral cavity, and the genital and gastrointestinal tracts of
humans and animals (Hammes & Vogel, 1995; Vaughan
et al., 2002). Lactobacilli have been recognized as potential
health beneficial microorganism in the human gastrointest-
inal tract, which is clearly reflected by the probiotic products
that are currently being marketed. A broadly accepted
definition of ‘probiotics,’ formulated by the World Health
Organization, states that probiotics are ‘live microorganisms
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which when administered in adequate amounts confer a
health benefit on the host’ (FAO/WHO, 2002). Notably, under
this definition, the endogenous intestinal tract bacteria are not
considered as probiotics unless they are isolated, cultured and
subsequently administered to the host. Although this defini-
tion specifies neither the mode of application nor the site of
action within the host body, the most common probiotic
applications use oral administration (mostly as fresh fermen-
tation products or dried bacterial supplements) and are
proposed to provide their health benefits through interactions
within the gastrointestinal tract. The continuing growth of
markets addressing health and well-being for consumers has
strongly stimulated molecular research into the metabolic
behavior and potential health beneficial effects of lactobacilli,
including (post)genomic research.
The extracellular characteristics of different lactobacilli are
of great importance for their capacity to interact with and
influence different factors encountered within the gastroin-
testinal tract (for reviews, see Lebeer et al., 2008; Kleerebezem
& Vaughan, 2009). This review focuses on the genome-based
prediction of the extracellular proteome of lactobacilli and the
comparative analysis of their genes and proteins. In addition,
it addresses the nonproteinaceous building blocks of the
Lactobacillus cell wall because they play a key role in interac-
tions with the host. We also discuss the current state of our
knowledge of the molecular interaction of specific extracellu-
lar components of lactobacilli with the host intestinal system,
combined with a short overview of postgenomic in vivo
approaches to unravel host responses to lactobacilli.
Genomics of lactobacilli
Following the initial focus of bacterial genomics on patho-
genic and paradigm laboratory species, the focus has shifted
to encompass many industrially relevant and benign bacteria,
including lactobacilli. The current public databases contain 18
complete Lactobacillus genomes, while at least 50 Lactobacillus
genome sequencing projects are ongoing at present (http://
www.ncbi.nlm.nih.gov/genomes/lproks.cgi). Extensive com-
parative analyses of the Lactobacillus (and other LAB) gen-
omes already revealed the molecular basis for some
phylogenetic, phenotypic and ecological diversities of the
different species encompassed within the genus (Canchaya
et al., 2006; Makarova et al., 2006; Cleasson et al., 2007;
O’Sullivan et al., 2009). In general, Lactobacillus genome
annotation and metabolic reconstruction revealed a consider-
able degree of auxotrophy for amino acids and/or other
cellular building blocks. Lactobacilli appear to compensate
for these metabolic ‘gaps’ by encoding a large variety of
import functions to incorporate environmental nutrients into
their metabolism. Niche-specific genomic adaptations are
clearly reflected within the Lactobacillus genomes. The typical
milk-adapted Lactobacillus bulgaricus and Lactobacillus helve-
ticus genomes (Makarova et al., 2006; Callanan et al., 2008) are
characterized by so-called genome decay and contain many
pseudogenes related to the utilization of several carbohy-
drates, reflecting their dedication to growth on lactose.
Notably, these characteristics are shared with Streptococcus
thermophilus, another LAB that is strongly adapted to the milk
habitat (Bolotin et al., 2004; Hols et al., 2005). In contrast, the
lactobacilli associated with the intestinal niche commonly
encode a large array of sugar import and utilization functions
(Kleerebezem et al., 2003; Makarova et al., 2006; Ventura et al.,
2009). Other functions that appear to be typically enriched in
intestinal lactobacilli include the (mucus binding) cell-surface
proteins and specific extracellular enzyme complexes that may
be involved in complex carbohydrate degradation (Boekhorst
et al., 2006a, b; Siezen et al., 2006). Analogously, the distribu-
tion of genes encoding bile salt hydrolase (BSH) among
lactobacilli, as well as a recent metagenomic study (Jones
et al., 2008), suggests a clear association of this function with
the intestinal habitat (Lambert et al., 2008a; O’Sullivan et al.,
2009). Such genes are essential for bile tolerance of Lactoba-
cillus plantarum and Lactobacillus salivarius (Lambert et al.,
2008b; Fang et al., 2009). The BSH-encoding gene has recently
been proposed to be an intestinal niche-specific molecular
marker for lactobacilli, as deduced from the detailed compar-
ison of three dairy, five intestinal and three multiniche
Lactobacillus genomes. Next to the bsh gene, the intestinal
lactobacilli appear to exclusively encode two specific sugar
transport functions, while the dairy lactobacilli exclusively
contain a set of six genes encoding functions related to
proteolytic capacities and restriction modification systems
(O’Sullivan et al., 2009). Analogously, a recent comparative
phylogenetic and single-gene marker study proposed reclassi-
fication of the Lactobacillus genus, and identified some key
taxonomic lactobacilli whose genome sequencing would
provide advanced molecular depth for such reclassification
(Claesson et al., 2008), and some of these are targeted by
ongoing whole-genome sequencing projects (http://www.
ncbi.nlm.nih.gov/genomes/lproks.cgi).
The current shift of bacterial genomics from single-strain
genomics to the pan-genomics of a species, including
postgenomic approaches such as comparative genome hy-
bridization using whole-genome DNA micro-arrays to as-
sess genomic diversity in relation to phenotypic diversity, is
illustrated by the lactobacilli L. plantarum (Molenaar et al.,
2005; Siezen et al., 2010) and Lactobacillus sakei (McLeod
et al., 2008). In addition, strain diversity can nowadays also
be addressed by the determination of multiple genome
sequences of individual isolates of a particular species (for a
review, see Tettelin & Feldblyum, 2009). For several Lacto-
bacillus species, currently, there are multiple genome se-
quences available, including L. plantarum, Lactobacillus
casei, Lactobacillus delbrueckii, Lactobacillus reuteri and
Lactobacillus rhamnosus, while some of the ongoing
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Lactobacillus genome sequencing projects target multiple
strains of a particular species, including six strains of
Lactobacillus crispatus and Lactobacillus jensenii (http://
www.ncbi.nlm.nih.gov/genomes/lproks.cgi). This trend is
bound to facilitate function assignment, including the
identification of potential probiotic ‘effector molecules’ as
has been illustrated for the mannose-specific adhesin func-
tion encoded by L. plantarum (Pretzer et al., 2005). Simi-
larly, comparative genomics may directly enable the
identification of strain-specific probiotic ‘effector mole-
cules.’ In this respect, the recent completion of the genome
sequence of the best-documented probiotic strain, L. rham-
nosus GG (Kankainen et al., 2009), and its comparison with
the closely related LC705 illustrates the potential of this
approach. The two L. rhamnosus genomes (both approxi-
mately 3.0 Mbp) display high levels of similarity and syn-
teny, but contain strain-specific genomic islands. The
genomic islands specific for strain GG encode approxi-
mately 80 proteins, including those involved in sugar
metabolism and transport, and exopolysaccharide biosynth-
esis. One of the L. rhamnosus GG-specific genome islands
encodes a pilin-like surface structure that is important in
adherence to intestinal mucus and is proposed to aid the
persistence of L. rhamnosus GG in vivo in the intestine
(Kankainen et al., 2009). Analogously, the genome of the
probiotic Lactobacillus johnsonii strain NCC533 is predicted
to encode fimbriae-like surface structures that may also play
a role in epithelial cell adhesion (Pridmore et al., 2004).
Genome mining aiming to identify probiotic effector
molecules is commonly focused on functions that are
targeted toward the cell surface, because these functions are
considered to be plausible candidates for probiotic interac-
tions with the intestinal system. As an example, in silico
exoproteome prediction for L. plantarum WCFS1 revealed at
least 12 proteins that are putatively involved in adherence to
host components such as collagen and mucin (Boekhorst
et al., 2006a). Mutational analysis of predicted extracellular
fibronectin and mucin-binding proteins of Lactobacillus
acidophilus NCFM confirmed their role in human epithelial
cell binding in vitro (Buck et al., 2005). Therefore, it is of
great importance that analysis of Lactobacillus genomes
includes the accurate predictions of surface-
associated functions, and encompasses the prediction of
subcellular location (SCL) and correlated membrane or
cell-wall anchoring mechanisms.
Lactobacillus protein transport pathways
Seven main protein secretion mechanisms have been char-
acterized in Gram-positive bacteria, namely the secretion
(Sec), twin-arginine translocation (Tat), flagella export
apparatus (FEA), fimbrilin-protein exporter (FPE), holin
(pore-forming), peptide-efflux ABC and the WXG100 secre-
tion system (Wss) pathways (for reviews, see van Wely et al.,
2001; Lee et al., 2006; Driessen & Nouwen, 2008; Desvaux
et al., 2009). These pathways are commonly conserved in
many Gram-positive bacteria, and by applying sequence
homology and protein-domain searches, we have evaluated
the presence of these protein secretion pathways in 13
published genomes of lactobacilli (Supporting Information,
Table S1). This targeted mining of the Lactobacillus genomes
revealed that these species do not encode the main factors
involved in the Tat, FEA and Wss protein secretion path-
ways, but do contain genes encoding the Sec, FPE, peptide-
efflux ABC and holin systems (Fig. 1; Table S1).
The major secretion pathway: Sec
The Sec translocase (Fig. 1) is the major system that
mediates protein transfer across the cytoplasmic membrane
in Gram-positive bacteria (for a review, see Driessen
& Nouwen, 2008). The translocase consists of a membrane-
embedded protein-conducting channel (SecYEG) and an
ATPase motor protein (SecA). The Sec translocase is usually
associated with the heterotrimeric complex SecDF-YajC,
which is involved in SecA activity regulation. The SecDF-
YajC may also bind to the YidC protein, which is relevant
for membrane insertion of integral membrane proteins
(Driessen & Nouwen, 2008). All Lactobacillus genomes
encode single copies of SecA, SecE, SecY, YajC and SecG
and double copies of YidC (Table S1), while no genes
encoding SecDF proteins could be found. In addition,
all Lactobacillus genomes encode single copies of the
components of the signal-recognition pathway, which is
involved in targeting of precursor proteins to the Sec
translocase, while the alternative signal-capturing pathways
depending on SecB (or its functional analogue in Bacillus
subtilis CsaA) appear to be absent in all Lactobacillus
genomes (Table S1).
All proteins targeted to the Sec translocase contain an N-
terminal signal peptide, which typically consists of three
regions: (1) the N region: a positively charged N terminus;
(2) the H region: a stretch of 15–25 hydrophobic residues;
and (3) the C region that may contain a signal peptidase
cleavage site (Driessen & Nouwen, 2008). During or after
translocation of the precursor protein across the cytoplas-
mic membrane, these signal peptides can be removed by
signal peptidases (SPases). Type-I SPase recognizes the
canonical AxAA cleavage site (van Roosmalen et al., 2004),
while Type-II SPase recognizes the L-x-x-C or the so-called
lipobox cleavage site (Sutcliffe & Harrington, 2002). All
Lactobacillus genomes encode a single Type-II SPase, while
the number of Type-I SPases ranged from one (in most
species) to three (in L. plantarum) (Table S1). This variable
number of Type-I SPases has also been found in other
Gram-positive genera (van Roosmalen et al., 2004).
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Holins
Holins (Wang et al., 2000) are small integral membrane
proteins that are primarily involved in the secretion of
muralytic enzymes that lack a signal peptide and play a
role in autolysis (Fig. 1). Holins are frequently encoded
by bacteriophage genomes, but can also be found in
Lactobacillus genomes. Identification of holins is hampered
by their low sequence similarity, but holins do share overall
structural and functional features that are commonly con-
served (Wang et al., 2000). Holins encoded within the
Lactobacillus genomes were identified on the basis of the
following criteria: (1) size range of 60–150 amino acids;
(2) at least one, but less than four transmembrane segments;
(3) a hydrophilic N terminus; (4) a polar, charge-rich
C-terminal domain; (5) reside in a gene context encoding
cell-lysis-associated proteins; (6) display at least 50% se-
quence similarity to known holin sequences; and/or (7)
harbor a holin-family domain. These analyses revealed that
holins are generally encoded by Lactobacillus genomes as
a part of the cell lysis system, although no holin could
be identified using these criteria in some Lactobacillus
strains (Table S1).
FPE
The FPE pathway (Fig. 1) is part of the competence
development (Com) pathway, allowing exogenous DNA
uptake across the bacterial cytoplasmic membrane (Chen &
Dubnau, 2004). The prepilin(-like) precursors involved in
this process are proposed to be translocated via a cleavage
event at the cytoplasmic side of the membrane by the
prepilin-specific SPase or transmethylase ComC (Chen &
Dubnau, 2004). In B. subtilis, the FPE system consists of
seven comG genes (comGA-GG operon) and a genetically
unlinked comC gene. These genes are involved in the
assembly of the pilin-like structure involved in DNA recog-
nition at the cell surface, including the export of the
prepilins ComGC-GE and GG and the DNA-binding surface
protein ComGF and the ComC-mediated prepilin cleavage
(Chen & Dubnau, 2004).
All Lactobacillus genomes have single copies of the
comGA-GC operon, and most species also have a comC
homologue (all except L. delbrueckii and Lactobacillus fer-
mentum), suggesting that the major constituents of the FPE
pathway are present in these lactobacilli. In addition,
L. delbrueckii ssp. bulgaricus American Type Culture Collec-
tion (ATCC) 111842 appears to encode an additional
ComGD prepilin, while L. delbrueckii ssp. bulgaricus ATCC
BAA-365 and Lactobacillus brevis ATCC 367 encode a
ComGF homologue. Besides the FPE pathway, single copies
of comE and comF genes, which are also involved in the
DNA-uptake process (Chen & Dubnau, 2004), were also
identified in all lactobacilli, except L. delbrueckii ssp. bulgar-
icus ATCC BAA-365 (Table S1).
Fig. 1. Schematic representation of the secretion systems and the final destination of the secreted proteins in Lactobacillus (the figure was adapted
from Desvaux et al., 2009). The secreted proteins (colored blue) can be grouped by their SCL as: (1) lipid anchored to the cytoplasmic membrane; (2)
attached to the cell wall either covalently (e.g. LPxTG proteins) or noncovalently (e.g. by exhibiting LysM, SLH or WXL domains/motifs); (3) anchored to
the cytoplasmic membrane via the N- or the C-terminal transmembrane helix, (4) released into the extracellular medium via Sec, holin or ABC
transporters; and (5) being part of cell-surface appendages, such as the competence pseudo-pili (assembled via FPE). ‘SP’ indicates that the proteins
carry an N-terminal signal peptide and their route targeting to the cytoplasmic membrane is depicted as black arrows, whereas the proteins lacking such
a signal peptide are shown by blue arrows. Secretion is depicted as red arrows and the integral membrane proteins (IMP) integration process is indicated
by violet arrows.
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Peptide efflux ABC transporters
Specific ABC transporter (Fig. 1) subfamilies that are
predominantly involved in export of antimicrobial peptide
(e.g. lantibiotics, bacteriocins and competence peptides)
(Havarstein et al., 1995) are capable of exporting proteinac-
eous substrates. For example, ABC exporters are responsible
for bacteriocin secretion in L. acidophilus (Dobson et al.,
2007) and L. plantarum (Diep et al., 1996). Using the
bacteriocin predictor BAGEL (de Jong et al., 2006), we
identified 3–12 putative bacteriocins in each Lactobacillus
genome. Most of the genes encoding predicted bacteriocins
appear to be genetically linked to genes encoding ABC
exporters, supporting the notion that peptide export via
ABC exporters can be commonly found in lactobacilli.
Lactobacillus exoproteome prediction
The term ‘secretome’ has been used to encompass compo-
nents of the translocation systems and their protein substrates
(Desvaux et al., 2009). However, in this review, we prefer to
use the term ‘exoproteome’ to only encompass the chromoso-
mal gene products that are transported across the Lactobacillus
cytoplasmic membrane, including molecules that become
surface localized, are parts of surface appendages or are
released into the environment (Greenbaum et al., 2001). To
enable a comprehensive overview of extracellular protein
components of the lactobacilli, our exoproteome definition
excludes integral membrane proteins with multiple mem-
brane-spanning regions (i.e. transport proteins, sensor ki-
nases, etc.), although it is clear that this extensive group of
Lactobacillus proteins may expose significantly sized domains
to the extracellular environment and play a key role in
bacterial interaction with the environment.
Dedicated efforts to predict the secretome/exoproteome
of individual Lactobacillus species have been reported before
(e.g. L. plantarum WCFS1; Boekhorst et al., 2006a). To
predict the SCL for each of the proteins encoded by the
lactobacilli (Table 1), the integrated SCL prediction pipeline
provided by LocateP (Zhou et al., 2008) was used. To date,
LocateP is the only SCL-pipeline that has successfully dealt
with the separation problem of the N-terminally anchored
proteins and the truly secreted proteins (defined as proteins
with a cleaved signal peptide that are released from the
bacterial cell), by incorporating a novel HMM-based N-
terminal anchor recognition system into the prediction
pipeline, which improved the accuracy of the differentiation
of these two groups of proteins to approximately 90% (Zhou
et al., 2008).
The predicted Lactobacillus exoproteomes contained two
main groups of proteins: the secreted proteins that are
released from the bacterial cell and the surface-associated
proteins. The latter group could be divided into several
subcategories based on different binding mechanisms: (1)
proteins that are anchored in the cytoplasmic membrane via
a single hydrophobic N- or C-terminal domain; (2) lipid-
anchored proteins (lipoproteins) that are N-terminally
anchored to long-chain fatty acids of the membrane; (3)
proteins covalently anchored to the peptidoglycan via a C-
terminal LPxTG motif; and (4) proteins noncovalently
bound to the cell surface by various binding domains or
Table 1. Predicted number of extracellular proteins encoded by 13 Lactobacillus genomes
Lactobacillus species and strains
Genome
size (kbp)
Total
number
proteins
Total
extracellular
proteins
SCL CWA
A B C D E F G H I J K L M
L. acidophilus NCFM 1993 1862 214 5 10 41 54 93 12 14 1
L. brevis ATCC 367 2291 2185 239 2 3 16 27 74 105 12 7 4 1 1
L. casei ATCC 334 2895 2751 306 4 11 18 46 46 160 19 9 5 1 2
L. delbrueckii bulgaricus
ATCC 11842
1865 1562 150 1 3 11 25 41 67 2 1 2
L. delbrueckii bulgaricus
ATCC-BAA-365
1857 1721 167 1 6 15 22 42 80 2 1
L. fermentum IFO3956 2099 1843 128 1 10 1 13 32 66 5 1 6
L. gasseri ATCC 33323 1894 1755 146 1 3 4 31 16 79 12 1 1 1
L. helveticus DPC4571 2081 1610 149 3 2 22 37 83 3 12 1 1
L. johnsonii NCC533 1993 1821 172 2 9 5 38 17 85 16 1 1 1
L. plantarum WCFS1 3308 3007 313 6 10 10 47 57 149 27 21 11 1 5
L. reuteri DSM20016 2000 1900 117 10 5 14 31 80 5 8 8
L. sakei 23K 1885 1879 178 2 3 13 27 45 83 3 14 4 1
L. salivarius UCC118 1827 1717 172 4 3 11 17 32 101 3 4 7
The SCL of these proteins and the number of proteins with cell wall anchoring (CWA) domains is predicted (including pseudogenes, but excluding
plasmids encoded genes).
SCL: A, C-terminally anchored; B, secreted via minor pathways (bacteriocin-like) (no CS�); C, N-terminally anchored (with CS�); D, lipid-anchored; E,
secreted (released) (with CS�); F, N-terminally anchored (no CS�); CS�, cleavage site.
CWA: G, LPxTG cell-wall anchor; H, choline binding domain; I, S-layer protein domain; J, WxL domain; K, LysM domain; L, peptidoglycan-binding
domain; M, SH3 domain.
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attached to other cell-wall protein(s) via protein–protein
interactions (Fig. 1). Several of the cell-wall-binding do-
mains that have been described were searched in the
Lactobacillus proteins (Table 1) that were predicted to be
secreted according to LocateP (which already includes a
search engine for LPxTG anchoring motifs).
Covalently anchored proteins
N- or C-terminally anchored proteins
The N-terminal signal peptides that target proteins to the
Sec translocation pathway contain the characteristic N, H
and C regions. During or after completion of the Sec-
dependent translocation process, the C region becomes
exposed to the extra-cytoplasmic side of the membrane.
Provided that the C region contains a Type-I or a Type-II
SPase target sequence, the signal peptide can be cleaved and
the mature protein is then released. However, many of the C
regions of Sec-translocated proteins do not possess this
cleavage motif [or contain a motif similar to the Type-I
motif that is not cleaved (Zhou et al., 2008)] and will remain
N-terminally anchored in the cell membrane. Many of the
proteins that are predicted to be N-terminally anchored
contain typical extracellular domains or functionalities and
their location at the extracellular side of the cell membrane
is highly plausible. In Lactobacillus genomes, the N-termin-
ally anchored proteins constitute the largest group of
membrane-anchored proteins (Table 1). These proteins are
mainly involved in extracellular bio-processes such as trans-
port, cell-envelope metabolism, competence, signal trans-
duction and protein turnover (Table S2).
In case a signal peptide C region contains a typical Type-I
SPase cleavage site and is thus processed, it may still be
anchored within the cytoplasmic membrane by a C-terminal
transmembrane domain, thereby exposing the mature do-
main to the extracellular side of the membrane. Lactobacillus
genomes encode a variable number of C-terminally an-
chored proteins, many of which have no known function
(Table S2).
Lipoproteins
Lipoproteins are the second largest membrane-anchored
group in the predicted Lactobacillus exoproteomes (Table
1). These proteins possess a signal peptide and are trans-
ported via the Sec pathway. The lipoprotein signal peptides
also contain the characteristic N, H and C domains,
although the H region is shorter than that in the Type-I
signal []peptides (Sutcliffe & Harrington, 2002) and the C
region contains the lipobox motif [L-(A/S)-(A/G)-C] that
directs them to the lipoprotein biogenesis machinery after
transport (Hutchings et al., 2009). The covalent binding of
the lipoprotein is generally achieved via diacylglyceryl
modification of the indispensable Cys-residue in the lipobox
by the lipoprotein diacylglyceryl transferase. Following
lipidation, cleavage occurs N-terminal of the Cys-residue
by the Type-II SPase, thereby anchoring the mature protein
to the membrane via thioether linkage (Hutchings et al.,
2009). The 13–47 lipoproteins predicted to be encoded by
the Lactobacillus genomes mainly encompass the substrate-
binding proteins of ABC transporters, but also some pro-
teins that are involved in adhesion, antibiotic resistance,
sensory processes, cell-envelope homeostasis and protein
secretion, folding and translocation (Table S2).
LPxTG-anchored proteins
A well-studied family of proteins that is covalently attached
to the peptidoglycan by the activity of the sortase (SrtA)
enzyme is characterized by the C-terminal LPxTG (based on
the main conserved residues) cell-wall-sorting motif (Boe-
khorst et al., 2005; Marraffini et al., 2006). LPxTG-contain-
ing proteins typically contain an N-terminal signal sequence
that contains a Type-I SPase cleavage site in its C region. The
LPxTG motif is located in the C-terminal region of the
mature domain and is followed by a C-terminal membrane
anchor domain, consisting of a stretch of hydrophobic
residues and a positively charged tail (Marraffini et al.,
2006). The sortase (SrtA) enzyme is a transpeptidase that
recognizes the LPxTG motif, cleaves the motif between the T
and G residues and covalently attaches the threonine car-
boxyl group to the peptidoglycan (Marraffini et al., 2006).
The Lactobacillus genomes encode a single copy of the
sortase (SrtA) and a variable number of LPxTG-motif
containing proteins, ranging from two proteins in L. del-
brueckii bulgaricus ATCC-BAA-365 and ATCC 11842 and to
27 proteins in L. plantarum WCFS1 (Table 1; Table S2).
Although there is some species-specific variation in the
amino acids of the LPxTG motifs (Boekhorst et al., 2005),
most of the sortase substrates could be readily detected in
the Lactobacillus genomes using the HMM from Boekhorst
et al. (2005) and have the conserved composition of the
motif (Table S2).
Noncovalent cell-wall-binding domaindetection
Domains that have been described to be involved in cell-wall
binding were searched using the Pfam database (http://pfam.
sanger.ac.uk/) and the protein sequences identified in this way
were inspected manually to verify their accurate detection.
LysM domains
The LysM (lysin motif) domain (Pfam PF01476) has been
found in many extracellular enzymes that are involved in
bacterial cell-wall metabolism, and is suggested to confer a
FEMS Microbiol Rev 34 (2010) 199–230c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
204 M. Kleerebezem et al.
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general peptidoglycan-binding function (Buist et al., 2008).
In all Lactobacillus genomes studied here, extracellular
proteins were found that contain at least one LysM domain
and almost all of these proteins perform cell-wall-related
enzymatic functions, in agreement with the proposed role of
LysM in peptidoglycan binding (Table 1).
Choline-binding domains
The choline-binding domains (Pfam PF01473) are a stretch of
20 amino acids that include multiple conserved tandem copies
of aromatic residues and glycines. They are mainly found in
extracellular enzymes such as autolysins and muramidases,
and are able to bind to choline residues of cell-wall teichoic
and lipoteichoic acids (LTA), thereby anchoring the protein to
the cell surface (Wren, 1991). In Lactobacillus, these choline-
binding domains appear to be present only in L. reuteri,
L. fermentum and L. salivarius (Table 1).
Putative peptidoglycan-binding domains
Another peptidoglycan-binding domain is composed of
three a helices located at the N or the C terminus of cell-
wall-degrading enzymes (Pfam PF01471). A single extracel-
lular protein containing this domain was found in
L. plantarum, L. johnsonii, L. casei, L. brevis, L. helveticus
and L. gasseri (Table 1).
S-layer proteins with SLH domains
S-layer proteins can form a paracrystalline monolayer that
coats the surface of bacteria, and are believed to be relevant
to cell-wall polysaccharide pyruvylation (Mesnage et al.,
2000; Avall-Jaaskelainen & Palva, 2005). In recent years, a
number of S-layer proteins have been identified experimen-
tally in L. acidophilus (especially the major S-layer protein
SlpA), L. helveticus and L. brevis (Avall-Jaaskelainen & Palva,
2005; Hollmann et al., 2007; Goh et al., 2009; Vilen et al.,
2009). The Pfam database contains different HMMs that
correspond to S-layer protein domains responsible for
noncovalent anchoring to the cell wall (SLAP or PF03217,
SLH or PF00395, S_layer_C or PF05124 and S_layer_N or
PF05123). Several putative S-layer proteins were found in
the genomes of L. acidophilus (14 proteins) and L. helveticus
(12 proteins), while a single protein was identified in
L. delbrueckii ssp. bulgaricus ATCC 11842, but not in
L. delbrueckii ssp. bulgaricus ATCC-BAA-365 using Pfam
and homology searches (Table 1).
WxL domains
The C-terminal cell-wall-binding domain designated WxL
was first identified in proteins of Lactobacillus and other
LAB based on in silico analysis (Chaillou et al., 2005;
Boekhorst et al., 2006a; Siezen et al., 2006). WxL
domain-containing proteins were found in gene clusters
that also encode additional extracellular proteins with C-
terminal membrane anchors and LPxTG-type peptidogly-
can anchors, suggesting that they form an extracellular
protein complex (Siezen et al., 2006). Recently, this domain
has been shown to be responsible for noncovalent interac-
tions between certain extracellular proteins and the bacterial
cell wall in Enterococcus faecalis (Brinster et al., 2007). In the
Lactobacillus exoproteomes, in total, 51 proteins containing
a WxL domain were identified, supporting an interaction of
these proteins with the peptidoglycan layer via their protein
C terminus (Table 1).
SH3 domains
The prokaryotic counterparts (SH3b) of the eukaryotic SH3
domains have been proposed to be involved in targeting and
binding to the peptidoglycan layer and are thought to
recognize specific sequences within the cross-linking peptide
bridges (Baba & Schneewind, 1996; Lu et al., 2006; Xu et al.,
2009). A search of the Lactobacillus genomes for these SH3b
domains identified several proteins in some lactobacilli
(Table 1). These proteins appear to function predominantly
in cell wall turnover.
Comparative exoproteomics ofLactobacillus
In total, 2451 putative extracellular proteins of 13 Lactoba-
cillus genomes were extracted from the LocateP-generated
database (Table 1, details in Table S2). The largest predicted
exoproteomes are found in L. casei (306 proteins) and
L. plantarum (313 proteins), and represent 11.1% and 10.4
% of all proteins encoded in these genomes, respectively.
The smallest exoproteomes were predicted for L. fermentum
(128 proteins, 6.9%) and L. reuteri (117 proteins, 6.1 %).
The most frequently found subcellular localizations of
proteins in these predicted exoproteomes are N-terminal
anchoring and secreted proteins, while the smallest category
is the C-terminally anchored proteins (Table 1). On average,
the functions of up to 60% of these extracellular proteins are
unknown. The proteins with a known (putative) function
are mostly involved in processes related to cell-envelope
metabolism, cell division, transport, competence, signal
transduction, protein turnover, exopolysaccharides bio-
synthesis, secretion, signaling/regulation and extracellular
enzymatic or binding functions.
Clustering of all proteins from 12 genomes of nonpatho-
genic lactic acid bacteria from the order Lactobacillales
(including 15 119 proteins from six completed Lactobacillus
genomes) using the method of clusters of orthologous
groups of proteins (COGs) resulted in 3465 Lactobacillales-
specific orthologous protein clusters (LaCOGs)(Makarova
et al., 2006; Makarova & Koonin, 2007). These LaCOGs
FEMS Microbiol Rev 34 (2010) 199–230 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
205The extracellular biology of lactobacilli
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included 1335 putative secreted proteins from Lactobacillus,
distributed over 338 orthology clusters. We have recently
extended these existing LaCOGs with the exoproteomes of 18
newly published LAB genomes (including seven new Lacto-
bacillus genomes) using BLASTP (Altschul et al., 1990), Inpar-
anoid (O’Brien et al., 2005) and in-house protein clustering
algorithms (Zhou et al., unpublished data), and stored all
information in the LAB-Exoproteome database (http://www.
cmbi.ru.nl/lab_exoproteome/). Here, we restrict our analysis
to the LaCOG information relevant for the predicted Lacto-
bacillus exoproteomes (see also Tables S2 and S3).
The predicted exoproteins in lactobacilli were clustered
into the 338 LaCOGs, placing approximately 76 % of the
total exoproteome into these orthologous groups. In most of
the clusters, the majority of the member proteins have
identical predicted SCLs and similar functionalities (includ-
ing 209 LaCOGs of conserved hypothetical proteins). Clus-
ters with known functions are mainly involved in typical cell
wall- or surface-associated functionalities (Table S3). A total
of 28 orthologous groups were found to be conserved in
all Lactobacillus genomes, and include, for example, the
housekeeping protease HtrA (LaCOG01440) and proteins
involved in cell-wall biosynthesis [LaCOG00243, penicillin-
binding protein (PBP) 2B], cell division (LaCOG01506,
cell shape-determining protein MreC) and competence
(LaCOG00097, DNA-entry nuclease) (Tables S2 and S3).
Conserved clusters represented within the majority of the
Lactobacillus genomes consist of extracellular enzymes, such
as carboxy-terminal proteinase (LaCOG01825), ATP synthase
(LaCOG01172), Zn-dependent protease (LaCOG01979) and
linoleic acid isomerase (LaCOG00663). Moreover, multiple
homologous proteins from one genome are found in some of
these LaCOGs, such as the four different members of the cell-
wall proteinase Prt family (LaCOG 90024) in L. casei, and the
four paralogous genes for cell-surface hydrolases (La-
COG01138) in L. plantarum. These distributions of LaCOG
representative proteins provide important insights toward
understanding the molecular evolution, diversity, function
and adaptation of the lactobacilli to specific environments
(Makarova et al., 2006; Makarova & Koonin, 2007). An
example is provided by the LaCOG distribution of the
mucus-binding proteins in Lactobacillus genomes. In total,
47 proteins with mucus-binding domain(s) were found in the
exoproteomes of six Lactobacillus genomes, distributed over
six separate LaCOGs. The largest cluster, LaCOG 01470,
contains 14 proteins that possess either the MucBP (Pfam
PF06458) domain or the recently defined extended mucus-
binding domain MUB (Boekhorst et al., 2006b), or both
domains. LaCOG00885 contains proteins that have only
MucBP domains. In LaCOG 01470, most proteins contain a
YSIRK signal peptide in their N terminus, which is a typical
characteristic of the gut L. acidophilus group lactobacilli (Bae
& Schneewind, 2003; Boekhorst et al., 2006b). The mucus-
binding proteins in group LaCOG00885 contain no YSIRK
signal peptide, and the cluster contains only proteins from the
typical plant lactobacilli L. plantarum and L. brevis (Fig. 2).
Lactobacillus cell-wall molecular biology
The Lactobacillus exoproteomes contain a variety of proteins
that are proposed to be anchored (covalently or noncova-
lently) to the basic components of the bacterial cell wall, such
as peptidoglycan, teichoic acid or polysaccharide. In addition,
these basic cell-wall components play an essential role in
communication mechanisms with the host environment
encountered within the gastrointestinal tract, including direct
signaling with the host tissues. These notions support a more
extensive review of the Lactobacillus cell-wall building blocks.
Peptidoglycan
In lactobacilli, like in all eubacteria, peptidoglycan is an
essential and specific cell-wall polymer found outside of the
cytoplasmic envelope. Its main function is to preserve cell
integrity from internal turgor pressure, which is of the order
of 20 atm in Gram-positive bacteria. In addition, peptido-
glycan is an important determinant of cell shape and serves
as a scaffold for the covalent anchoring of other cell-wall
polymers, wall teichoic acids (WTA) and wall polysacchar-
ides (WPS), and some surface proteins (Fig. 3) (Delcour
et al., 1999; Vollmer et al., 2008a).
Peptidoglycan is composed of glycan strands consisting
in their unmodified form of alternating residues of b-1-
4-linked N-acetyl muramic acid (MurNAc) and N-acetyl-
glucosamine (GlcNAc) cross-linked by short peptides. The
D-lactoyl residue of the MurNAc is substituted by a penta-
peptide ending in D-Ala-D-Ala or pentadepsipeptide ending
in D-Ala-D-Lac (D-Lac, D-lactate), whose composition in
lactobacilli in its unmodified form is L-Ala(1)-g-D-Glu(2)-(L-
Lys or meso-A2pm or L-Orn)(3)-D-Ala(4)-(D-Ala or D-Lac)(5)
[2,6 diaminopimelate (A2pm); ornithine (Orn)] (Kandler &
Weiss, 1986; Delcour et al., 1999; Lebeer et al., 2008). Many
modifications of this basic composition are found in the
glycan strands and its associated stem peptides (Fig. 3).
In lactobacilli, N-deacetylation of GlcNAc/MurNAc
(L. fermentum) and 6-O-acetylation of MurNAc (L. plantar-
um, L. casei, L. acidophilus and L. fermentum) of glycan
strands has been reported (Fig. 3) (Delcour et al., 1999;
Vollmer, 2008; E. Bernard, unpublished data). Both mod-
ifications play important roles in the physiology of Gram-
positive bacteria and in their interactions with their hosts,
such as an increased resistance to lysozyme that could help
to escape the innate immune system (for a recent review, see
Vollmer, 2008). Besides the well-recognized resistance to
lysozyme of lactobacilli, the functional role of these
two modifications has not yet been investigated in this
group. In silico analysis of complete genome sequences of
FEMS Microbiol Rev 34 (2010) 199–230c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
206 M. Kleerebezem et al.
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12 Lactobacillus species (Table 2) revealed the absence of a
GlcNAc deacetylase gene (pgdA) in most lactobacilli,
while nearly all of them contain at least one copy of a
putative MurNAc O-acetyltransferase gene (oatA) (Table 2).
Notably, two paralogues were found in L. plantarum WCFS1
and L. sakei 23K, which suggest an important role of O-
acetylation in these two species. On the other hand, the
absence of oatA in L. delbrueckii ssp. bulgaricus ATCC 11842
suggests a lack of importance of this function in the
milk niche. Preliminary results of the analysis of an
OatA-deficient strain of L. plantarum WCFS1 confirmed its
contribution to lysozyme resistance (E. Bernard & P.A.
Bron, unpublished data). The contribution of O-acetylation
to the recognition of peptidoglycan fragments by host
receptors [Toll-like receptors (TLR), nucleotide-binding
oligomerization domain proteins (NOD)] and/or escape of
innate immune defenses remains to be investigated in
lactobacilli.
Variations in the composition, cross-linking and postmo-
difications of stem peptides in lactobacilli are mostly present
at positions 2, 3 and 5. In most lactobacilli, an L-Lys residue
is found in position 3 connected to a D-Asp included in the
cross bridge (L-Lys-D-Asp type) of two adjacent stem pep-
tides, but L-Orn-D-Asp or meso-A2pm direct types of linkage
are also found (e.g. in L. fermentum or L. plantarum,
respectively) (Fig. 3 and Table 2) (Schleifer & Kandler,
1972; Kandler & Weiss, 1986). The D-Asp residue is gener-
ated from L-Asp by an aspartate racemase (RacD) before its
ligation to L-Lys by a recently identified ligase of the ATP-
grasp family (AslA) (Bellais et al., 2006; Veiga et al., 2006).
Orthologues of RacD and AslA are present in all genomes of
lactobacilli known to contain a peptidoglycan of L-lys-D-asp
or Orn-D-Asp types, while AslA is remarkably absent in
L. plantarum (meso-A2pm direct) (Table 2). Surprisingly,
L. plantarum contains an racD orthologue, suggesting
that aspartate racemase could be required for a metabolic
Fig. 2. The different architectures of some
Lactobacillus mucus-binding proteins (distributed
in two LaCOGs).
FEMS Microbiol Rev 34 (2010) 199–230 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
207The extracellular biology of lactobacilli
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function other than peptidoglycan biosynthesis in this
species. In terms of postmodifications, amidations of D-Glu
at position 2 (yielding D-iso-Gln), of meso-A2pm and of D-
Asp (yielding D-iso-Asn) have been identified in L. casei or
L. plantarum (Fig. 3) (Billot-Klein et al., 1997; Asong et al.,
2009). However, little is known about the functional role of
these amidations in lactobacilli. In Lactococcus lactis, the
asnH gene encoding asparagine synthase was recently shown
to be responsible for D-Asp amidation, and the deficiency of
D-Asp amidation in this species resulted in an increased
sensitivity to cationic antimicrobials, and affects the activity
of endogenous autolysins (Veiga et al., 2009). Orthologues
of asnH were detected in all genome sequences of lactobacilli
with the L-Lys-D-Asp type, suggesting that amidation of
D-Asp is a general feature in lactobacilli. Intriguingly, an
asnH orthologue was also found in L. plantarum WCFS1
(meso-A2pm direct), which could be involved in amidation
of a residue of the stem peptide other than D-Asp, possibly
meso-A2pm or D-Glu (E. Bernard, unpublished data). No-
tably, most of these modifications (meso-A2pm vs. L-Lys,
amidation of D-Glu and meso-A2pm) of stem peptides of
peptidoglycan fragments affect recognition by the host
receptors [e.g. NOD1, NOD2, peptidoglycan recognition
proteins (PGRPs), TLR2] of the host innate immune system
(illustrated for NOD1 and NOD2 in Fig. 3) (Girardin et al.,
2003; Roychowdhury et al., 2005; Wolfert et al., 2007; Asong
et al., 2009). For example, amidations of meso-A2pm and
D-Glu of L. plantarum were shown to modulate TLR2
recognition (Asong et al., 2009). These variations among
lactobacilli could significantly impact on their immunomo-
dulatory properties and thus affect their probiotic function.
A remarkable feature of many lactobacilli is their intrinsic
resistance to the glycopeptide vancomycin (VanR) (Table 2).
In L. plantarum and L. casei, where vancomycin resistance
(VanR) has been investigated, this antibiotic resistance is the
result of the 100% incorporation of D-Lac instead of D-Ala at
position 5 of the stem peptide (Fig. 3) (Ferain et al., 1996;
Billot-Klein et al., 1997; Delcour et al., 1999; Goffin et al.,
2005; Deghorain et al., 2007). The D-Ala-D-Lac terminus has
a 1000-fold decreased affinity for vancomycin compared
with the D-Ala-D-Ala terminus. In enterococci, vancomycin
resistance by D-Ala/D-Lac substitution is acquired in most
cases by the transfer of a large transposon (e.g. Tn1546),
encoding the van genes responsible for the reprogramming
of the biosynthesis of peptidoglycan precursors [for a recent
review, see Mainardi et al., 2008). By contrast, the lactoba-
cilli that are intrinsically resistant to vancomycin produce
D-Lac in variable amounts as an end-product of fermenta-
tion (Table 2). However, this feature is also found in
vancomycin-sensitive species (e.g. L. helveticus, L. delbrueck-
ii ssp. bulgaricus). A D-lactate dehydrogenase gene (ldhD) or
a D-hydroxyisocaproate dehydrogenase gene (hicD) is pre-
sent in nearly all lactobacilli, the latter being responsible for
the production of a low amount of D-Lac in the vancomy-
cin-resistant L. casei species (Viana et al., 2005). D-Lac
production can also be achieved from the conversion of
L-Lac into D-Lac by a lactate racemase (lar operon) as shown
recently for D-Lac production in L. plantarum (Goffin et al.,
2005). The lar operon is also present in L. brevis,
L. fermentum and L. sakei (Table 2). The lack of an
identifiable ldhD gene in L. sakei suggests that lactate
racemization is the only route for D-Lac production (Mal-
leret et al., 1998). In L. plantarum and L. sakei, altering D-Lac
production results in the loss of the VanR phenotype (Goffin
et al., 2005; P. Hols, unpublished data). Furthermore, in
L. plantarum, complete abolition of D-Lac production
(ldhD, lar double mutant) results in a growth arrest that
can be fully restored by external D-Lac supplementation or
Fig. 3. Schematic representation of the
peptidoglycan structure of lactobacilli and modes
of action of PGHs. (a) Peptidoglycan structure of
Lactobacillus casei illustrating an L-Lys-D-Asp
cross bridge. (b) Peptidoglycan structure of
Lactobacillus plantarum with a meso-A2pm direct
cross bridge. The localizations of the different
postmodifications are indicated: WPS/WTA
anchoring on MurNAc (Mur), amidations (�NH2)
of the stem peptide, O-acetylation (�O-Ac) of
MurNAc (Mur) and N-deacetylation (�NH2) of
MurNAc and GlcNAc (Glc). Molecular patterns
recognized by NOD1 and NOD2 are boxed by
dashed and plain lines, respectively. Cleavage
sites of PGHs are indicated by arrows: A,
N-acetylmuramoyl-L-alanine amidase; E,
endopeptidases; G, glucosaminidase; LT, lytic
transglycosylase; and M, muramidase.
FEMS Microbiol Rev 34 (2010) 199–230c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
208 M. Kleerebezem et al.
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Tab
le2.
Spec
ific
feat
ure
sof
pep
tidogly
can
and
TAan
dth
eir
asso
ciat
edgen
esin
12
com
ple
tegen
om
ese
quen
ces
of
lact
obac
illi
L.ac
idophilu
s
NC
FM
L.bre
vis
ATC
C367
L.ca
sei
ATC
C334
L.del
bru
ecki
i
ATC
C11842
L.fe
rmen
tum
IFO
3956
L.gas
seri
ATC
C33323
L.hel
veticu
s
DPC
4571
L.jo
hnso
nii
NC
C533
L.pla
nta
rum
WC
FS1
L.re
ute
ri
DSM
20016
L.sa
kei
23K
L.sa
livar
ius
UC
C118
Peptidogly
can
6-O
-ace
tyla
tion�
1?
1?
1?
??
1?
??
oat
A1
11
1�
w�
w1
11
11
11
oat
A2
��
��
��
��
1�
1�
N-d
eace
tyla
tion�
??
??
1?
??
??
??
pgdA
�1
��
��
��
��
��
Cro
ss-lin
king
type�
Lys-
D-A
spLy
s-D-A
spLy
s-D-A
spLy
s-D-A
spO
rn-D
-Asp
Lys-
D-A
spLy
s-D-A
spLy
s-D-A
spm
A2pm
-direc
tLy
s-D-A
spLy
s-D-A
spLy
s-D-A
sp
aslA
11
11
11
11
�1
11
racD
11
11
11
11
11
11
asnH
11
11
11
11
1(a
snB1/2
)1
11
Van
Rphen
oty
pe�
SR
RS
RS
SS
R?
RR
D-lac
pro
duct
ion�
L/D
L/D
L(D)
DL/
DL/
DL/
DL/
DL/
DL/
DL/
DL(
D)
ldhD
(hic
D)
11
hic
D1
11
11
11
�hic
D
lar
oper
on
�1
��
1�
��
1�
1�
ddl(
Y-
or
F-ty
pe)
YF
FY
FY
YY
FF
FF
Teic
hoic
acid
s
WTA
type�
Gro
?G
ro?
�G
ro-P
??
Gro
??
Gro
-P/R
bo-P
??
?
tag
gen
es1
1�
1�
11
11
�1
1
LTA
type�
Gro
?G
ro?
Gro
-PG
ro-P
Gro
-P?
Gro
-PG
ro-P
Gro
-PG
ro-P
??
ltaS
11
11
11
11
11
11
1
ltaS
21
11
11
1�
�w
11
�1
D-a
lanyl
ated
TA�
??
11
??
11
11
??
dlt
oper
on
11
11
11
11
11
11
� Spec
ific
feat
ure
sor
phen
oty
pe
asre
port
edin
the
liter
ature
for
the
spec
ies.
w Var
iable
among
gen
om
esse
quen
ces
of
the
sam
esp
ecie
s.
1,p
rese
nce
;�
,abse
nce
;?,
unav
aila
ble
oruncl
ear;
S,se
nsi
tive
;R,r
esis
tant;
L/D,r
acem
icm
ixtu
reof
L-an
dD-L
ac;L
(D),
smal
lam
ountof
D-L
ac;D
,exc
lusi
vepro
duct
ion
of
D-L
ac;G
ro?,
TAco
nta
inin
ggly
cero
l.
FEMS Microbiol Rev 34 (2010) 199–230 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
209The extracellular biology of lactobacilli
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only partially by expressing a D-Ala-D-Ala-forming ligase
(Goffin et al., 2005). In this species, the cell-wall biosynthesis
machinery is specifically dedicated to the production of
D-Lac-ended peptidoglycan precursors. Mutation analysis
has shown that the specificity of the Ddl ligases for either
D-Ala-D-Ala or D-Ala-D-Lac is associated with either a
phenylalanine (F type) or a tyrosine (Y type), respectively,
at a specific position in the enzyme [position 216 in DdlB of
Escherichia coli (F type; Park et al., 1996) and 261 in Ddl of
Leuconostoc mesenteroides (Y type; Park & Walsh, 1997)].
Interestingly, Ddl enzymes from all VanR lactobacilli are of
the F type, while the VanS species possess Y-type enzymes
(Table 2). This observation strongly suggests that vancomy-
cin resistance in lactobacilli takes place principally by a
reprogramming of the biosynthesis of peptidoglycan pre-
cursors. The vancomycin resistance among lactobacilli may
reflect the selective advantage of this phenotype in niches
that also contain glycopeptide antibiotic producers, which
may especially hold true for Lactobacillus species with a
broader niche specificity or those that are associated with
plant fermentations such as L. plantarum, L. brevis, L. casei
and L. fermentum.
Peptidoglycan is continuously remodeled during growth
by the action of a variety of peptidoglycan hydrolases
(PGH). These enzymes are involved in separation of daugh-
ter cells, peptidoglycan turnover and autolysis in the sta-
tionary phase. They are also involved in many other
processes such as adhesion, biofilm formation, resuscitation
of dormant cells or allolysis in genetic transformation (for a
recent review, see Vollmer et al., 2008b). Through autolysis
in the host and cell-wall turnover, lactobacilli could release
muramyl-peptides that are known to interact with receptors
of the immune system. For instance, muramyl-peptides
from L. plantarum ATCC 8014 display immunoadjuvant
activity, but the in vivo role of peptidoglycan fragments of
lactobacilli remains largely unexplored (Kotani et al., 1975).
In silico analysis of the PGH content of lactobacilli shows
that besides low-molecular-weight PBPs (carboxypepti-
dases) that are mainly involved in peptidoglycan matura-
tion, they display a variety of PGH, from 14 members in
L. acidophilus to 26 in L. reuteri (Layec et al., 2008). These
PGH are distributed into four classes: N-acetyl-glucosamini-
dases/-muramidases and lytic transglycosylases hydrolyzing
the glycan strands; N-acetylmuramoyl-L-alanine amidases
separating the stem peptides from the glycan strands; and
endopeptidases of the NLPC/P60 or CHAP families hydro-
lyzing a range of bonds of the cross-linked stem peptides
(Fig. 3) (Layec et al., 2008; Vollmer et al., 2008b). A more
detailed examination of the PGH complement (16 genes) of
L. plantarum WCFS1 revealed a high level of redundancy in
lytic transglycosylases (six members), glucosaminidases/
muramidases (five members) and NLPC/P60 endopepti-
dases (four members), while a single L-alanine amidase is
present (Table 3). Redundancy in these three classes is a
general feature in lactobacilli, with some variations such as
an overrepresentation of lytic transglycosylases in L. plantar-
um and endopeptidases in L. reuteri. A recent systematic
inactivation of nine PGHs of L. plantarum WCFS1 shows
that the inactivation of only two [lp_2645 (acm2) glucosa-
minidase/muramidase and lp_3421 endopeptidase] has a
significant impact on cell morphology (T. Rolain, unpub-
lished data). These two PGH and the endopeptidase lp_2162
were recently identified as cell-wall-associated proteins of
L. plantarum using a proteomic approach, reinforcing their
functional role in this species (Beck et al., 2009). Remark-
ably, 12 out of 16 PGH of L. plantarum display a modular
organization, where the catalytic domain is associated with a
peptidoglycan-binding domain (one to five SH3 motifs or
one to two LysM motifs) and systematically to a domain rich
in alanine, serine and threonine (AST motif) (Table 3). This
last domain is suspected to be glycosylated, but its func-
tional role is unexplored. The modular organization of PGH
in lactobacilli is a general feature, because at least seven types
of domains in addition to LysM and SH3 have been
identified (for a recent review, see Layec et al., 2008). In
addition to cell-wall binding and targeting PGH to their site
of action, these domains could fulfill other biological func-
tions such as adhesion by binding to receptors on eukaryotic
cells (Layec et al., 2008; Vollmer et al., 2008b). Besides the
functional role of Acm2 from L. plantarum WCFS1 in cell
separation (Palumbo et al., 2006) and the endopeptidase
activity of the S-layer protein of L. acidophilus ATCC 4356
(Prado et al., 2008), this important class of exoenzymes is
poorly characterized in lactobacilli.
To conclude, small variations in the composition and
modifications of peptidoglycan, as well as the endogenous
capacity to release muramyl-peptides among lactobacilli, are
strongly suggested to be important in host–microorganism
interactions and adaptation to the ecological niche. Future
work aiming to modulate the fine structure of peptidoglycan
and/or the content of PGH could help to better understand
these important roles.
Teichoic acids (TA)
Besides peptidoglycan, the cell wall of lactobacilli comprises
TA, which are essential anionic polymers of Gram-positive
bacteria and represent up to 50% of the cell-wall dry weight.
TA are involved in various aspects of the functionality of the
cell wall. Together with peptidoglycan, they form a poly-
anionic matrix or gel contributing to the porosity, elasticity
and electrostatic steering of the cell envelope. They are also
involved in cation homeostasis, in particular of Mg21 and
protons, the latter being important in the maintenance of a
proton gradient in the cell wall. Among a large range of
identified biological functions, TA participate in the
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modulation of the activity of PGHs, the binding of surface
proteins, phage adsorption, cell adhesion and interaction
with the immune system (counterpart of Gram-negative
lipopolysaccharide) (Delcour et al., 1999; Neuhaus &
Baddiley, 2003).
Lactobacilli deserve specific mention in terms of TA since
they were initially discovered by Baddiley and colleagues in
L. plantarum (formerly Lactobacillus arabinosus) (Baddiley,
1989). Most lactobacilli investigated for their TA content
possess two types of anionic polymers: WTA that are
covalently bound to MurNAc of peptidoglycan glycan
strands via a linkage unit (Fig. 3) and LTA that are anchored
on the cytoplasmic membrane through a glycolipid, but that
are also found to be loosely bound to peptidoglycan and
even released into the extracellular medium. Both TA types
are decorated by D-alanyl esters associated or not with
glycosyl (mainly glucose) substitutions (Sharpe et al., 1964;
Kandler & Weiss, 1986; Fischer et al., 1990; Delcour et al.,
1999; Neuhaus & Baddiley, 2003; Lebeer et al., 2008).
The characterized WTA of lactobacilli are generally com-
posed of polyglycerophosphate [poly(Gro-P)], but with
some variations such as the presence of polyribitolpho-
sphate [poly(Rbo-P)] in around half of the strains of
L. plantarum (e.g. ATCC 8014) (Tomita et al., 2008, 2009)
or a complete absence in L. casei (Kandler & Weiss,
1986)(Table 2). Although the genetic determinants of WTA
biosynthesis have not been investigated in lactobacilli, the
tag and tar genes and their products responsible for poly
(Gro-P) and poly(Rbo-P) WTAs, respectively, have been
characterized extensively in B. subtilis and Staphylococcus
aureus (Lazarevic et al., 2002; Brown et al., 2008). Although
initially reported as essential, depletion of WTA was recently
achieved in both species by inactivation of the gene encod-
ing the first enzyme of its biosynthetic pathway (D’Elia et al.,
2006a, b). Nevertheless, WTA play a critical role in cell-shape
maintenance in B. subtilis and Mg21 dependence for growth
(Schirner et al., 2009). Orthologues of tagO/tarO are present
in most species, with the remarkable exceptions of L. casei,
which was previously reported to be WTA deficient and also
in L. fermentum and L. reuteri. In these three species, all the
tag/tar orthologues are completely absent, showing that the
presence of this secondary anionic polymer is a variable
feature among lactobacilli and suggesting that LTA in these
species is sufficient to confer all the important biological
functions of TA. Among lactobacilli, WTA ultrastructures of
L. plantarum are the best characterized including the nature
of the linkage unit (Kojima et al., 1985; Tomita et al., 2008,
2009). The WTA structure in this species was reinvestigated
recently by nuclear magnetic resonance (NMR), and the
monomeric units of poly(Gro-P)/poly(Rbo-P) WTAs were
shown to be decorated not only by D-alanyl substitutions but
also by multiple glucose residues in a range of configura-
tions, including kojibiose and one or two glucose interca-
lated in the main chain of poly(Gro-P) (at least nine
different types) (Tomita et al., 2008, 2009). These analyses
revealed a high structural diversity in L. plantarum, suggest-
ing that the WTA structure is important for its lifestyle.
Although the in silico annotation of tag/tar genes is compli-
cated due to similarities between the different transferases/
polymerases, L. plantarum WCFS1 contains all the necessary
tag/tar genes, which are scattered in eight different loci, in
contrast to B. Subtilis, where all tag or tar genes are clustered
Table 3. In silico analysis of the PGH content in Lactobacillus plantarum WCFS1
Gene Family Size (aa) SS CBD AST domain LaCOG
acm2 (lp_2645) Glucoaminidase/muramidase 785 1� 5 SH3 1
lp_3093 860 1 5 SH3 1
acm3 (N-M-C)w 612 1 3 SH3 1
acm1 (lp_1138) 213 1 � � 00918
lys (lp_1158) 258 1 � � 01725
lytH (lp_1982) L-Ala amidase 282 1 1 SH3 � 01848
lp_3421 Endopeptidase Nlpc/P60 370 1� 1 LysM 1 90015
lp_2162 496 1� 2 LysM 1 90015
lp_2520 297 1 � � 00646
lp_1242 243 1 � �lp_0302 Lytic transglycosylase 267 1 1 LysM 1 01094
lp_3014 204 1 1 LysM 1 01094
lp_3015 220 1 1 LysM 1 01094
lp_0304 Lytic transglycosylase (WY domain) 212 1 1 LysM 1 01589
lp_2845 314 1 1 LysM 1 01589
lp_2847 354 1 1 LysM 1 01589
�Shown as cell-wall associated (Beck et al., 2009).wPseudogene in three fragments (N-M-C).
1, presence; � , absence; SS, signal sequence; CBD, cell-wall biding domain.
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together (Lazarevic et al., 2002). The redundancy of some
genes (e.g. two putative tagD and tagF, three putative tagB
and for some a higher similarity to tar genes) suggests that
L. plantarum has the genetic content to produce both types
of WTAs. Furthermore, six genes code for putative glucosyl-
transferases similar to TagE, which was shown in B. subtilis
to be responsible for WTA glucosylation (Honeyman &
Stewart, 1989). Besides a role for glucosylation of WTA in
phage recognition in L. plantarum, the functional role of
WTA and its substitutions remains elusive in lactobacilli
(Delcour et al., 1999).
In terms of the structure of LTA of lactobacilli, all isolated
polymers are composed of poly(Gro-P), which are deco-
rated at least by D-alanyl esters (Sharpe et al., 1964; Kandler
& Weiss, 1986; Fischer et al., 1990; Delcour et al., 1999;
Neuhaus & Baddiley, 2003). Until recently, no gene was
identified that could specifically abolish the biosynthesis of
this important cell-wall constituent. The recent discovery
of the LTA synthase LtaS [poly(Gro-P) polymerase] in
S. aureus and B. subtilis (four paralogues) and their inactiva-
tion allowed specific blocking of LTA biosynthesis (Grund-
ling & Schneewind, 2007; Schirner et al., 2009). The
LtaS-deficient mutants of both species are viable, but display
several defects, including a strongly altered cell morphology
and problems in septa positions in S. aureus and a lack of
control of cell elongation and separation in B. subtilis
(Grundling & Schneewind, 2007; Oku et al., 2009; Schirner
et al., 2009). The latter morphological defect is proposed to
result from a modified Mg21/proton homeostasis, which
affects enzymes of peptidoglycan biosynthesis and the role
of LTA in the control of PGHs (Schirner et al., 2009). The
lethality of a double depletion of WTA and LTA in both
species shows that at least one anionic polymer is essential
for cell survival (Oku et al., 2009; Schirner et al., 2009). All
complete genomes of lactobacilli contain at least one copy of
ltaS, with a second copy identified in most species (Table 2).
In contrast to the apparent variability of WTA synthesis
among lactobacilli, the conservation of at least one ltaS gene
suggests that these species produce LTA consistently.
The LTA structure of four Lactobacillus strains (L. plantarum
WCFS1, L. rhamnosus GG, L. reuteri 100-23 and L. del-
brueckii ssp. lactis ATCC 15808) was investigated recently
by NMR (Palumbo et al., 2006; Perea Velez et al., 2007;
Walter et al., 2007; Raisanen et al., 2007; Schirner et al.,
2009). The chains comprise 20–22 Gro-P in L. plantarum
and L. reuteri, and 33 and 50 residues in L. delbrueckii and
L. rhamnosus, respectively. D-Alanyl esters are unique sub-
stituents in L. plantarum (42% D-Ala : GroP) and L. rham-
nosus (74% D-Ala : GroP), and are associated with a low level
of glucosyl residues in L. reuteri (76% D-Ala : GroP and 6%
glc : GroP) and L. delbrueckii (24% D-Ala : GroP and 3%
glc : GroP). This detailed analysis shows that LTA of lactoba-
cilli are variable in chain length, percentage and composi-
tion of substitutions, and possibly in the nature of the
lipid anchor.
D-Alanyl esters of LTA (and WTA) strongly contribute to
the function of TA because the positively charged amino
groups partially counteract the negative charges of the
backbone phosphate groups. D-Alanylation of TA is per-
formed by at least four proteins encoded in the dlt operon.
The biochemistry of D-alanylation was principally studied by
Neuhaus and colleagues using L. rhamnosus 7469 as a model
(for an extensive review, see Neuhaus & Baddiley, 2003). The
importance of D-alanyl esters in lactobacilli is reinforced by
the presence of a dlt operon in all complete genomes of
lactobacilli (Table 2). The impact of a depletion of D-alanyl
esters of TA was investigated in three of the four strains cited
above by the inactivation of the dlt operon (Palumbo et al.,
2006; Perea Velez et al., 2007; Walter et al., 2007); the dlt
mutants had either strongly reduced or no D-alanyl esters,
but the chain length was, respectively, threefold increased in
a subpopulation of LTA of L. plantarum WCFS1, 1.7- and 8-
fold reduced in the two subpopulations of LTA of
L. rhamnosus GG and unaffected in L. reuteri 100-23.
Interestingly, the level of glucosylation was increased from
undetectable in LTA of the wild-type L. plantarum WCFS1 to
24% substitution in the dlt mutant. A fivefold increase was
also observed in L. reuteri 100-23, but no change could be
detected in L. rhamnosus GG. In the latter, a modification of
the lipid anchor was reported. These modifications suggest
that D-alanylation contributes directly or indirectly to the
chemical composition of LTA in these three species. The
more negatively charged TA in the absence of D-alanyl esters
results in an increased sensitivity to positively charged
antimicrobial peptides (e.g. nisin) in all three mutants. An
increased sensitivity to human b-defensin-2 is also reported
for L. rhamnosus GG. This suggests that the positive charges
of D-alanyl esters in lactobacilli contribute to the general
defense against positively charged antimicrobial molecules.
The depletion of D-alanyl esters also results in elongated cells
in L. plantarum and L. rhamnosus, a defect in cell separation
in L. rhamnosus, perforations of the cell wall at division sites
in L. plantarum, an increased autolysis in the three mutants
and a strongly affected growth in L. plantarum. All these
phenotypic modifications suggest that the machinery of
peptidoglycan assembly/degradation is affected, probably
due to a charge modification affecting the proton gradient,
homeostasis of cations and the control of positively charged
PGHs. The Acm2 PGH of L. plantarum, involved in cell
separation, was shown to participate to this pleiotropic
phenotype (Palumbo et al., 2006). Acid tolerance is strongly
reduced in the mutants. Thus, in L. reuteri and L. Rhamno-
sus, growth at a low pH was affected, and a higher sensitivity
to gastric juice challenge was reported for L. rhamnosus, but
the resistance to acidic conditions per se appeared to be
unchanged in L. reuteri. In L. reuteri, which is a true resident
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212 M. Kleerebezem et al.
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of the rodent gut and forms a biofilm in the forestomach of
Lactobacillus-free mice, the depletion of D-alanyl esters
strongly impairs gut colonization and the formation of
biofilms in the forestomach. Mutant cells present in the
mouse forestomach display damaged cell envelope struc-
tures, showing that the high level of D-alanylation protects
this species under hostile conditions prevailing in the rodent
forestomach (Walter et al., 2007). In L. johnsonii La1, cell-
surface-associated LTA is important for adhesion to human
enterocyte-like Caco-2 cells, possibly through hydrophobic
interactions (Granato et al., 1999). In L plantarum WCFS1,
D-alanyl esters modulate specific immune responses (Gran-
gette et al., 2005; Rigaux et al., 2009). Notably, the dlt
mutant is more protective compared with the wild-type
strain in a mouse model of colitis. In vitro, the mutant
induces a dramatically increased level of the anti-inflamma-
tory cytokine IL-10 in peripheral blood monocytes. The
immunomodulation effect of purified LTA of both wild type
and mutant is largely TLR-2-dependent (Grangette et al.,
2005). However, this interesting impact of a variation in the
LTA composition observed with the L. plantarum mutant
seems species-specific, because the dlt mutant of L. rhamno-
sus is not affected compared with the wild type in similar
in vitro immune modulation experiments (Perea Velez et al.,
2007). In addition, the expression of the dlt operon of
L. plantarum WCFS1 was modulated in the mouse cecum
following a modification of the diet, showing that this
species adapts its level of D-alanylation in vivo in response
to environmental conditions (Marco et al., 2009).
Overall, TA of lactobacilli contribute to many aspects of
the extracellular characteristics of lactobacilli and affect
many phenotypic traits, including cell adhesion, biofilm
formation, survival under hostile conditions and interaction
with the immune system. In the future, the selective
complete depletion of WTA or LTA by gene inactivation in
lactobacilli would certainly help to better understand their
specific contribution in commensal–host interactions and
adaptation to this ecological niche.
Extracellular polysaccharides
Cell-wall polysaccharides are ubiquitous components of the
cell envelope of lactobacilli. These polysaccharides, which are
generally neutral, can be found to be covalently attached to
MurNAc of peptidoglycan glycan strands (WPS) (Fig. 3),
loosely associated with the cell envelope or released into the
extracellular medium (exopolysaccharides, EPS). When the
polysaccharides form a thick shell (capsule) closely associated
or bound to the cell envelope, they are generally named
capsular polysaccharides (CPS). However, the distinction
between WPS, EPS and CPS appears to be somewhat artificial
because the abundance and localization of polysaccharides is
for instance strongly dependent on the growth conditions
(Delcour et al., 1999; Lebeer et al., 2008). Polysaccharides of
lactobacilli are generally heteropolysaccharides of complex
structures differing in the nature of sugar monomers, the
modes of linkage, branching and substitutions. The following
sugar moieties are present in Lactobacillus polysaccharides:
glucose, galactose, rhamnose, GlcNAc, N-acetylgalactosa-
mine, Glucuronate and Gro-P, along with phosphate, acetyl
and pyruvyl modifications (for reviews, see De Vuyst &
Degeest, 1999; Lebeer et al., 2008). Polysaccharides biosyn-
thetic genes of lactobacilli are grouped into clusters (up to
25 kb). These clusters are highly variable in terms of glycosyl-
transferases responsible for the biosynthesis of the repetition
unit. This high variability of polysaccharides clusters from
strain to strain was recently revealed in the L. plantarum
species and in members of the L. acidophilus group using
microarray approaches (Molenaar et al., 2005; Berger et al.,
2007). The complexity of polysaccharides biosynthesis is even
increased by the presence of multiple polysaccharides gene
clusters per strain, for example, four gene clusters were
identified in the genome of L. plantarum WCFS1.
To date, polysaccharides of lactobacilli have been shown
to be involved in phage absorption in L. plantarum and
L. casei, in the attachment of the S layer in L. buchneri and in
immunomodulation using for instance polysaccharides ex-
tracted from L. kefiranofaciens and L. rhamnosus RW-9595M
(for reviews, see Delcour et al., 1999; Lebeer et al., 2008).
Recently, knockouts of polysaccharides geneclusters have
been reported in L. casei Shirota, L. johnsonii NCC533 and
L. rhamnosus GG (Denou et al., 2008; Yasuda et al., 2008;
Lebeer et al., 2009). In L. casei Shirota, the inactivation of 8
(cps1A to J) out of 10 genes included in a polysaccharides
gene cluster led to a large reduction of a high-molecular-
mass polysaccharides in the cell-wall fraction. In vitro
experiments performed with heat-killed cells on mouse
macrophage cell lines suggest a role of this polysaccharides
as a relevant immune suppressive modulator of macrophage
activation (Yasuda et al., 2008). In L. johnsonii NCC533,
deletion of the entire polysaccharides gene cluster eliminates
a fuzzy layer decorating the outer rim of the cell wall.
Interestingly, this mutant has a slightly increased persistence
time in mice, suggesting that surface polysaccharides could
hinder other cell adhesions (Denou et al., 2008). Similarly,
the inactivation of the priming glycosyltransferase gene welE
of one of the polysaccharides gene clusters of L. rhamnosus
GG, responsible for the biosynthesis of long galactose-rich
polysaccharides, results in increases in adherence to epithe-
lial cells and mucus and in biofilm formation. A compensa-
tory mechanism is present because the mutant shows an
increase in the amount and length of a second glucose-rich
surface polysaccharides as indicated by atomic force micro-
scopy studies using functionalized tips with lectins (Francius
et al., 2008; Francius et al., 2009; Lebeer et al., 2009).
Interestingly, electron microscopy of mutant cells not only
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revealed the absence of the polysaccharide layer but also an
increased exposure of fimbriae or pili (Lebeer et al., 2009).
Fimbrial genes have been reported in L. johnsonii NCC533,
but they are uncommon among lactobacilli, and the direct
visualization of pili on Lactobacillus cells has only been
shown for L. rhamnosus GG (Kankainen et al., 2009), where
they become more prominently exposed upon removal of
the polysaccharides.
Although the above studies all revealed the importance of
polysaccharides of lactobacilli in commensal–host interac-
tions, much remains to be learned about biosynthetic
pathways, regulation, locations, compositions, size and con-
formation of polysaccharides in lactobacilli. Moreover, the
apparent biological diversity of polysaccharides among
Lactobacillus species and strains, combined with its potential
role in host–microorganism interaction, supports further
research on these interesting molecules.
Extracellular function in adaptation to thehost environment
In vitro approaches
Several biological barriers are encountered by bacteria dur-
ing residence in and travel through the different parts of the
host’s gastrointestinal tract (Fig. 4), such as gastric acidity
encountered in the stomach, bile salt and digestive enzyme
challenges in the duodenum, a relatively high osmolarity in
the colon, as well as stress conditions associated with oxygen
gradients that are steep at the mucosal surface, while the
colonic lumen is virtually anoxic. Moreover, considerable
bacterial competition is encountered throughout the gastro-
intestinal tract and is most severe in the colon where cell
numbers are the highest (Zoetendal et al., 2006a). Several
studies describe the in vitro response of intestinal bacteria to
a simplified model that mimics (components of) the stress
encountered in the host’s gastrointestinal tract (for a review,
see Lebeer et al., 2008). However, these simplified systems
fail to accurately address the physiochemical or the micro-
bial complexity of the intestinal environment in vivo (de Vos
et al., 2004; Kleerebezem & Vaughan, 2009). Nevertheless,
some common responses are apparent, including activation
of DNA/protein protection and repair mechanisms, differ-
ential regulation of two-component and other regulatory
mechanisms and induction of systems for the active removal
of acid and bile-related stress compounds (Corcoran et al.,
2008; Lebeer et al., 2008).
One particularly relevant aspect in light of this review is
the fact that many lactobacilli appear to modify the different
macromolecules that constitute the cell envelope, thereby
contributing to maintenance of cell integrity during the
in vitro exposure to a variety of gastrointestinal-tract-relevant
stress conditions (Lebeer et al., 2008). For example, lower pH
and bile salts influence the fatty acid and phospholipid
composition in the cell membranes of L. casei (Fozo et al.,
2004) and L. reuteri (Taranto et al., 2003). Moreover, Tween
addition to the growth medium of L. rhamnosus, Lactobacillus
paracasei and L. salivarius results in up to a 3-log increased
survival during exposure to gastric juice. A 55-fold higher
oleic acid content in the fatty acid composition of
L. rhamnosus GG led to the suggestion that the resultant
more rigid structure caused by increased membrane fatty acid
saturation levels may explain the observed enhanced survival
characteristics (Corcoran et al., 2007).
The genetic factors important during low pH and bile
stress have also been investigated in several lactobacilli. Acid
shock induces several genes potentially involved in mem-
brane fluidity regulation or peptidoglycan biosynthesis and
Fig. 4. Challenges encountered by lactobacilli during travel through the gastrointestinal tract. The bacterial population sizes indicate the entire
microbiota densities at the different locations in the human intestine. Scanning electron microscopy (SEM) insets display Lactobacillus plantarum WCFS1
cultures after in vitro growth with and without bile salts (left and right, respectively), exemplifying the severe physiological consequences of one of the
stresses encountered by lactobacilli in the intestine.
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214 M. Kleerebezem et al.
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organization in L. reuteri ATCC 55730, including a putative
phosphatidyl glycerophosphatase and a putative esterase
gene, encoding a paralogue of PBPs (Wall et al., 2007).
Mutation of the latter gene causes increased sensitivity to
both gastric juice (Wall et al., 2007) and bile exposure
(Whitehead et al., 2008). Similarly, several genes potentially
involved in cell-envelope and surface protein biosynthesis
are induced by bile exposure of L. acidophilus NCFM (Pfeiler
et al., 2007) or L. plantarum WCFS1 (Bron et al., 2004b,
2006). Furthermore, L. acidophilus gene disruption mutants
in a cell-division protein (cdpA) and surface layer protein A
(slpA) have an increased bile resistance and reduced osmo-
tolerance (Altermann et al., 2004; Klaenhammer et al.,
2005), further highlighting the importance of subtle mod-
ifications in cell-envelope composition in the resilience of
LAB to persist under different stress conditions relevant for
gastrointestinal-tract survival.
In vivo approaches
Several postgenomics approaches have addressed the mole-
cular adaptation of lactobacilli to the intestinal environment
using animal model systems, for example differential pro-
moter activity studies focusing on four L. casei promoters
revealed that this LAB is metabolically active in the mouse
intestine and initiates de novo protein synthesis to adapt to
this niche (Oozeer et al., 2005). Furthermore, two genome-
wide genetic screens in L. reuteri (Walter et al., 2003) and
L. plantarum (Bron et al., 2004a) exploiting in vivo expres-
sion technology revealed the induction of 3 and 72 genes in
the mouse intestinal tract, respectively. Strikingly, the
L. plantarum protein encoded by lp_2718 is a homologue
of the only conserved hypothetical protein that was identi-
fied as being in vivo induced (ivi) in L. reuteri (Walter et al.,
2003; Bron et al., 2004a). A subsequent dedicated mutagen-
esis approach demonstrated that the ivi gene, encoding a
methionine sulfoxide reductase B, contributes to the ecolo-
gical performance of L. reuteri in the murine gut (Walter
et al., 2005). The L. plantarum ivi genes identified include
four predicted extracellular proteins (lp_0141, lp_0800,
lp_1403 and lp_2940), several sugar PTS transport systems
and a copper-transporting ATPase (copA) (Bron et al.,
2004a). Subsequent qRT-PCR analysis unraveled the spatial
and temporal in vivo expression patterns of a subset of the
identified ivi genes (Marco et al., 2007). Furthermore, a
dedicated mutagenesis approach underlined the critical
contribution of lp_2940 and copA to murine gut persistence
(Bron et al., 2007).
More recently, technical advances have allowed the isola-
tion of high-quality bacterial RNA derived from intestinal
samples (Zoetendal et al., 2006b). The targeted studies
described above were followed by transcriptome approaches
to describe bacterial behavior in the gastrointestinal tract.
One such approach revealed the expression of specific sets of
genes in L. johnsonii when it resides in different compart-
ments of the mouse gastrointestinal tract (Denou et al.,
2007). While no colon-specific genes were identified, the
induction of expression of specific sugar PTS transport
systems could be established in the jejunum, the stomach
and the cecum. Furthermore, the stomach-specific genes
include several multidrug transport systems, a cation efflux
protein, as well as a copper-transporting ATPase. This gene
induction pattern closely resembles the ivi genes identified
in L. plantarum using the in vivo expression technology
approach described above (Bron et al., 2004a; Denou et al.,
2007; Marco et al., 2007). Three genetic loci potentially
important for intestinal persistence have been identified by
correlating differences in murine intestine persistence of two
L. johnsonii strains with differential genome content and in
vivo transcriptome data, and subsequent mutagenesis de-
monstrated the importance in murine intestinal perfor-
mance of a mannose PTS system and a protein that shares
30% identity with immunoglobin A proteases from patho-
genic bacteria (Denou et al., 2008).
Recently, the transcriptomes of L. plantarum WCFS1 in
the cecum of mono-associated mice fed either a western-
style (high fat, low fiber) or a standard chow diet (low fat,
high fiber) were compared with in vitro transcriptomes
(Marco et al., 2009). Notably, 9 and 32 genes encoding cell
envelope- and wall-localized functions were induced in the
ceca of the chow- and western diet-fed mice, respectively.
These in vivo induced genes primarily encode putative cell-
wall-anchored and lipoproteins with unknown functions,
rather than polysaccharide, peptidoglycan or teichoic acid
biosynthetic capacities, illustrating the limited current
knowledge of the factors important for L. plantarum in this
niche. Five genes in this category are induced in vivo
independent of the dietary regime, including the cell-surface
complex operons cscI, cscVII and cscVIII (Siezen et al., 2006),
potentially facilitating the ability of L. plantarum to utilize
host or dietary glycans in the distal gut habitat (Marco et al.,
2009). The expression level of lp_2940 is induced in mice
irrespective of the dietary regime used, while lp_0800 is only
induced in mice fed a normal mouse chow diet, reiterating
the importance of these proteins for the in vivo fitness of
L. plantarum and illustrating the pronounced effect of diet
on in situ microbial responses. Moreover, the cation efflux
protein previously identified utilizing an in vivo expression
technology approach is upregulated in mice fed a western
diet, and induction of genes encoding functions involved in
carbohydrate transport and metabolism is the largest in-
duced functional category, independent of the mice dietary
regime (Marco et al., 2009).
All studies mentioned above exploit the murine gut as a
model for the human gastrointestinal system. However, one
recent study has determined the transcriptome profile of
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L. plantarum 299v, a strain closely related to WCFS1, in large
intestinal biopsies obtained from patients with possible
colon cancer who volunteered to participate in a probiotic
trial before surgery (de Vries et al., 2006). Subsequently, the
transcriptome profiles obtained were compared with over
100 in vitro transcriptomes available for L. plantarum
WCFS1. Like the mouse gastrointestinal transcriptome
datasets described above for L. plantarum WCFS1, the
in situ profiles for L. plantarum 299v in the human colon
suggest that this organism strongly adapts its capacity for
carbohydrate acquisition and cell-surface composition. The
L. plantarum response in all in vivo samples includes
upregulation of the capsular polysaccharide biosynthesis
operon cps3 and the cell-surface protein clusters cscI and
cscVIII (Marco et al., 2009; M.L. Marco, M. Wels, W.M. de
Vos, E.E. Vaughan & M. Kleerebezem, unpublished data).
Moreover, lp_0800 and lp_2940 were upregulated in the
human colon, corroborating the many observations suggest-
ing the importance of these genes in the intestine (Bron
et al., 2004a; Marco et al., 2007, 2009; M.L. Marco, M. Wels,
W.M. de Vos, E.E. Vaughan & M. Kleerebezem, unpublished
data). These overlapping responses for both L. plantarum
WCFS1 and 299v in different gastrointestinal compartments
using different mammalian model systems (mono-asso-
ciated mice fed a western or a chow diet, colonized mice
fed a chow diet and human volunteers) suggest a diet-, host-
and microbiota-independent core response in multiple
L. plantarum strains. Hence, the associated extracellular
molecules are robust key factors strongly affecting the (pro-
biotic) functionality of this LAB in the gastrointestinal tract.
Adhesion to mucus and host mucosa
The environmental adaptations described above can be
anticipated to contribute mainly to the in situ survival and/
or the persistence characteristics of the lactobacilli during
their passage through the intestinal tract. Another aspect
considered important for probiotics is the capacity to adhere
to mucosal surfaces and/or tissues. Therefore, a variety of
studies have specifically addressed the LAB extracellular
adhesins and their contribution in direct microbial interac-
tions with host cells or compounds. These studies typically
use in vitro models such as epithelial cell lines, immobilized
intestinal mucus or extracellular matrix molecules, includ-
ing collagen and fibronectin to determine adherence capa-
cities (Velez et al., 2007; Lebeer et al., 2008).
Several Lactobacillus extracellular compounds described
as important for adhesion appear to contribute in a rather
unspecific manner. For example, LTA provides the main
component of hydrophobicity to the Lactobacillus cell
envelope, which appears to be the reason for its involvement
in the adhesive characteristics of L. johnsonii (Granato et al.,
1999), L. rhamnosus (Lebeer et al., 2007) and L. reuteri
(Walter et al., 2007). Similarly, exopolysaccharides, which
contributes to cell-surface physiochemical properties and
shielding of other cell-surface adhesins, is important for the
adhesive characteristics of L. acidophilus (Lorca et al., 2002)
and L. rhamnosus (Ruas-Madiedo et al., 2006). Moreover,
the authors of several papers suggest that pleiotrophic effects
could contribute to the altered adhesive characteristics of
their constructed mutants. The L. acidophilus cdpA gene
encodes a cell-wall-modifying enzyme that promotes cell
division, and a mutant has reduced adhesion, which could
be explained by a loss of anchoring or translocation or
integrity of important adhesins (Altermann et al., 2004).
Genome mining for candidate extracellular adhesins
encoded by L. acidophilus led to the identification of five
proteins potentially involved in adhesion to epithelial cells
(Buck et al., 2005). Mutant studies confirmed the involve-
ment in adhesion to Caco2 cells in vitro, for three of the five
proteins selected (Mub, FbpA, SlpA) (Buck et al., 2005). The
authors argue that the observed phenotype of the
L. acidophilus slpA (encoding surface layer protein) mutant
is likely due to the loss of multiple surface proteins that may
be embedded in the S layer, whereas the contributions of
Mub and FbpA (encoding a mucin-binding and a fibronec-
tin-binding protein, respectively) to adhesion are more
likely to be via a specific interaction with epithelial cells.
Nevertheless, a similar role in adhesion to intestinal epithe-
lial cells could be established for the S-layer proteins in
L. brevis (Hynonen et al., 2002), L. crispatus (Toba et al., 1995;
Antikainen et al., 2002) and L. helveticus (Johnson-Henry
et al., 2007), whereas the L. brevis SlpA protein also mediates
adhesion to extracellular matrices such as fibronectin (Hyno-
nen et al., 2002). These variations to a theme surrounding the
surface layer proteins of lactobacilli, including variations in
surface layer-associated protein domains, support a relevant
role for these proteins in the interaction with the host
environment. Such a role in immune cell recognition was
recently exemplified for the L. acidophilus SlpA.
A homologue of the L. acidophilus Mub protein described
above, with 25% identity at the amino acid level, is present
in L. reuteri 1063 (Roos & Jonsson, 2002). The proteins are
of similar size and contain a similar number of mucus-
binding domains (MucBP domain; 17 and 14 domains,
respectively). Interestingly, intact L. reuteri cells can adhere
to pig and hen mucus. A direct role for Mub protein in
mucin binding was shown using purified fusion proteins
consisting of maltose-binding protein fused to various
MucBP domain-containing repeats (Roos & Jonsson,
2002). Another adhesin identified in L. reuteri (strain
NCIB11951) is the collagen-binding protein (CnBP), which
can adhere to solubilized Type-I collagen (Aleljung et al.,
1994). Notably, CnBP has sequence similarities to the
solute-binding domain of bacterial ABC transporters, a
domain also found in the mucus adhesion-promoting
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216 M. Kleerebezem et al.
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protein (MapA) that was reported to mediate the binding of
L. reuteri 104R to Caco-2 cells and mucus (Miyoshi et al.,
2006). The Mub proteins of L. acidophilus and L. reuteri
mentioned above contain all the required elements for
sortase-dependent anchoring to the cell wall. The L. salivar-
ius UCC118 genome was predicted to encode 10 sortase-
dependent extracellular proteins, and three of the encoding
genes were shown to be expressed in vitro (van Pijkeren
et al., 2006; Table 1). Mutation of either the lspA or the
sortase-encoding srtA gene in L. salivarius significantly
reduced the capacity to bind to HT-29 cells, whereas lspC
and lspD mutants adhered at similar levels as the wild-type
strain. Remarkably, lspA encodes a protein containing seven
MucBP domains (van Pijkeren et al., 2006), and this
repeated domain structure appears to be a feature shared
by many extracellular proteins that contain MUB or MucBP
domains (Fig. 2). The broad distribution of MUB- or
MucBP-domain-containing proteins suggests that they may
play a conserved role in intestinal adhesion in many
lactobacilli, which is corroborated by their relative enrich-
ment in species that are associated with the intestinal niche
(Fig. 2). However, it remains to be seen whether the
commonly used in vitro cell line models accurately mimic
the actual in vivo situation, where host defense systems,
competition with the resident microbiota, mucosal shed-
ding and peristaltic flow are likely to modify adhesion
(Lebeer et al., 2008). Notably, in this respect, sortase-
deficient mutants of both L. plantarum (Bron et al., 2004c;
Pretzer et al., 2005) and L. johnsonii (Denou et al., 2008)
display a wild-type persistence phenotype in the intestinal
tract of mice. These data further emphasize the importance
of experiments that would allow translation of the in vitro
adhesion data obtained so far to the actual in situ situation
in the gastrointestinal tract. A clear example for such
translation experiments was recently presented for the
L. rhamnosus GG pilin-like structures (Kankainen et al.,
2009). In view of the annotation of Lactobacillus exopro-
teomes and prediction of adherence capacities for individual
proteins, it is important to note that the nomenclature of the
binding domains mentioned above is debatable, because
their assignments have been primarily based on binding
ligands present in the in vitro assay in which they were
tested. The actual substrate specificity of the mucus-,
fibronectin-, collagen-, etc., binding domains remains to be
established, and they may recognize specific features (e.g.
attached glycosyl residues or other molecular features)
present in the currently assigned substrates rather than
particular features of the specific proteins per se.
Probiotic effector molecules
In contrast to the impressive amount of information on the
Lactobacillus adaptive response to the host gastrointestinal
environment, and the discovery of several adhesins, there is
very limited knowledge on the molecular mechanisms by
which probiotics exert their health-beneficial effects on the
host (Marco et al., 2006; Kleerebezem & Vaughan, 2009). To
date, only a few candidate probiotic effector molecules have
been discovered, and while for some there is convincing
evidence for their proposed role in vivo, some others still
require validation in situ.
Inhibition of intestinal pathogens
In densely populated niches, such as the gastrointestinal
tract, lactobacilli are in constant competition for nutrients
with each other and other bacteria. Within the framework of
probiotic applications, there is a growing interest in the use
of dietary lactobacilli for their potential for controlling the
gastrointestinal microbial ecosystem to support human
health (Corr et al., 2009). This possibility is exemplified by
the studies that support the suppressive effect of L. johnsonii
LA1 on Helicobacter pylori colonization (Gotteland &
Cruchet, 2003), or indicate the modulatory capacity of
L. acidophilus LB on H. pylori infection (Coconnier et al.,
1998). Several mechanisms for these suppressive effects of
lactobacilli toward pathogens have been suggested, includ-
ing competitive exclusion, immunomodulation, inhibition
of virulence expression and/or direct killing or inhibition by
antimicrobial peptides (Corr et al., 2009). The antimicrobial
peptides produced by LAB can be classified into different
classes and subclasses, encompassing the lantibiotics (Class
I) and the canonical double-Glycine leader bacteriocins
(Class II) (for reviews, see Klaenhammer, 1993; Kleerebezem
& Quadri, 2001; Eijsink et al., 2002). Although the majority
of the studies on LAB antimicrobial peptides primarily aim
at their possible application as preservative agents in food
products (Cotter et al., 2005), some studies also address
their potentially important role in intestinal competitiveness
and their potential relevance as effector molecules in vivo in
probiotic applications such as preventing pathogen infec-
tions or more general microbial control in this complex
ecosystem. A recently published study elegantly demon-
strated this role of bacteriocins at the molecular level (Corr
et al., 2007). Lactobacillus salivarius UCC118 produces
Abp118, a Class IIb bacteriocin that peaks in the early
stationary phase, a process regulated through quorum
sensing via the induction peptide AbpIP. Administration of
the Class IIb bacteriocin (Abp118) producing L. salivarius
UCC118 protected mice against oral infection with Listeria
monocytogenes, while mice that were administered an
Abp118-negative L. salivarius mutant were not protected
from listeriosis. Moreover, a direct linkage of the protective
effects to the Abp118 bacteriocin was proven by the com-
plete abolition of the protective effect of the Abp118-
producing L. salivarius strain when mice were infected with
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217The extracellular biology of lactobacilli
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a L. monocytogenes strain expressing the Abp118-immunity
protein, AbpIM. This study provides convincing evidence
for the potential of bacteriocins produced by lactobacilli and
other LAB to prevent diseases associated with intestinal
pathogens (Corr et al., 2007).
Induction of the plantaricin immunity protein PlnI
during murine gastrointestinal transit of L. plantarum,
observed using three independent approaches (Bron et al.,
2004a; Marco et al., 2007, 2009), suggests that bacteriocins
are also important for the in vivo performance of this
species. Moreover, preliminary observations suggest that
plantaricin-related functions are important in the capacity
of L. plantarum WCFS1 to modulate specific cytokine
responses in human blood-derived immune cells in vitro
(M. Meijerink, S. van Hemert, N. Taverne, M. Wels, P. de
Vos, H. Savelkoul, J. van Bilsen, M. Kleerebezem, P.A. Bron,
& J.M. Wells, unpublished data). These experiments further
emphasize the importance of bacteriocins in probiotic
function, which may involve multiple mechanisms, includ-
ing immunomodulation and direct killing of pathogens.
The competitive exclusion concept of probiotic function-
ing proposes that lactobacilli or other health benefit cultures
may adhere to epithelial sites that also function as site of
adherence of pathogenic bacteria, and thereby lactobacilli
may prevent the docking of pathogens onto epithelia and
may thus inhibit their infection efficacy. As an example,
certain L. plantarum strains have been reported to adhere
specifically to mannose-containing moieties present on hu-
man intestinal cells (Adlerberth et al., 1996), which is
proposed to competitively exclude adhesion of enterotoxi-
genic E. coli (ETEC) and thereby prevent infection. Using a
gene-trait matching approach (Molenaar et al., 2005; Pretzer
et al., 2005), the absence or the presence of the L. plantarum
WCFS1 lp_1229 gene could be correlated to the capability of
14 L. plantarum strains to agglutinate yeast, an in vitro assay
utilized to determine the mannose-specific adherence phe-
notype (Pretzer et al., 2005). Subsequently, an L. plantarum
lp_1229 mutant was found to have lost its ability to
agglutinate yeast and, therefore, lp_1229 was designated
msa (mannose-specific adhesin). The encoded protein con-
tains two potential MucBP domains and a SasA domain,
associated with a ConA-type lectin, further supporting a
role for Msa in carbohydrate recognition and binding
(Pretzer et al., 2005). Intriguingly, specific regions of the
Msa protein sequence vary in different L. plantarum strains;
the number of MucBP domains in the protein can range
from a single domain up to four or five domains, which may
be correlated to the difference observed for their quantita-
tive capacity to adhere to mannose or mannose polymers
(Gross et al., 2009). However, no in vivo experiments have
been reported that either establish or disprove the competi-
tive exclusion hypothesis and a potential role for Msa
therein. Nevertheless, studies using a pig model system
revealed that the msa mutant has a decreased association
with intestinal epithelia, and increased jejunal fluid absorp-
tion. Moreover, preliminary results indicate that expression
of the host pancreatitis-associated protein, a protein with
proposed bactericidal properties, is only induced by the
wild-type strain (Gross et al., 2008). Although these studies
do not directly support the competitive exclusion model,
they do show that specific adhesin capacities of lactobacilli
may affect mucosal biology in situ, which may strengthen
defense mechanisms in the host mucosa.
Mucosal integrity
Probiotic applications are also proposed to reinforce muco-
sal barrier function, by maintaining or supporting epithelial
integrity. The molecules of lactobacilli that may be involved
in this process remain largely unknown, and their function-
ing may involve complex host signaling pathways and
multiple mechanisms. Interestingly, two abundantly se-
creted proteins of L. rhamnosus GG, P40 and P75, were
recently purified and demonstrated to activate the Akt
pathway in epithelial cells. This pathway plays a central role
in promoting host cell survival by inactivation of several
proapoptotic pathways. In addition, P40 and P75 inhibited
cytokine-induced epithelial cell apoptosis, promoted cell
growth in human and mouse colon epithelial cells and
decreased tumor necrosis factor (TNF)-induced colon
epithelial damage (Yan et al., 2007; Seth et al., 2008).
Although these elegant in vitro studies illustrate the poten-
tial of these molecules to promote intestinal homeostasis
through specific signaling pathways and the prevention of,
or protection against damage, validation of their in vivo role
is still not available.
Immune system modulation
An important conceptual health benefit of probiotics is
based on their capacity to promote immunotolerance by
priming dendritic cells (DCs) to stimulate the differentia-
tion of IL-10 producing regulatory T-cells (Kleerebezem &
Vaughan, 2009). A recent paper supporting this concept
reports L. reuteri and L. casei strains that can interact with
the C-type lectin receptor DC-SIGN (DC-specific intercel-
lular adhesion molecule 3-grabbing nonintegrin), which
leads to the subsequent induction of regulatory T cells
(Smits et al., 2005). Moreover, an slpA mutant in
L. acidophilus, which was known to have a decreased capacity
to adhere to Caco-2 cells (Buck et al., 2005), also displayed
reduced binding to DC-SIGN and consequently led to a more
proinflammatory cytokine profile (Konstantinov et al., 2008).
SlpA was postulated to be the first probiotic bacterial DC-
SIGN ligand (Dsl) identified that is functionally involved
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218 M. Kleerebezem et al.
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in the modulation of DC and T-cells function. Interestingly,
the ConA (recognizing mannose) and AAL (specific for
a6 fucose) lectins can bind purified SlpA, and because
mannose and fucose are the glycans recognized by DC-
SIGN, these preliminary results have led the authors to
further investigate the carbohydrate moieties present on
different S-layer proteins (Konstantinov et al., 2008). As
already concluded above (see the section on noncovalent
anchoring motifs), only a few lactobacilli produce S-layer
proteins, implying that species that lack Slp proteins interact
with DC-SIGN via an alternative ligand. To this end, the
gene-trait matching approach enabled the identification of a
candidate Dsl in L. plantarum (Molenaar et al., 2005). Dsl is
predicted to be a C-terminally anchored extracellular pro-
tein, rich in threonine and serine residues. The overrepre-
sentation of these amino acids may suggest that this protein
could be O-glycosylated, which may be involved in the
postulated DC-SIGN recognition (D. Remus, M. Meijerink,
J.M. Wells, M.L. Marco, P.A. Bron, and M. Kleerebezem,
unpublished data). Protein glycosylation mechanisms and
their potential implications on the functionality of lactoba-
cilli are discussed below.
Lactobacillus glycoproteins?
Although protein glycosylation was long believed to be
restricted to Eukarya, it is now clear that bacteria can also
form protein-attached N-glycans via asparagine residues, as
well as O-glycans via threonine and/or serine residues
(Messner, 2004; Szymanski & Wren, 2005; Weerapana &
Imperiali, 2006; Abu-Qarn et al., 2008). Most studies on
bacterial protein glycosylation revealed specific O-glycan
attachment to one or two abundant polymeric surface
proteins such as flagellins, pilins and S-layer proteins
(Schaffer et al., 2001; Benz & Schmidt, 2002; Fletcher et al.,
2007). Moreover, a limited number of general glycosylation
systems have been described in bacteria, including the N-
glycosylation machinery in Campylobacter jejuni and related
species (Fig. 5b). This intestinal pathogen harbors the pgl
gene cluster, which encodes the PglFED enzymes responsible
for modifying the nucleotide-energized sugar UDP-GlcNaC
to di-N-acetylbacillosamine (Sharon, 2007). This modified
monosaccharide is attached to a phosphorylated undecapre-
nyl lipid carrier on the cytosolic side of the cell membrane by
PglC, one of the five glycosyltransferases also encoded in the
Fig. 5. Schematic representation of polysaccharide biosynthesis systems in different bacteria. (a) Displays the typical PS synthesis system of
Lactobacillus rhamnosus GG that is basically shared by other lactobacilli (although with different sugar compositions of the polysaccharides) and is
primarily involved in the production of a cell-wall-anchored polysaccharide (or exopolysaccharide in some other lactobacilli), but may play a role in
delivering repeating unit oligoscaccharides to a so far unknown transferase that can transfer these oligosaccharides to extracellular proteins. (b) Displays
the Campilobacter jejuni (and relative species) biosynthesis pathway, proposed for oligosaccharide production and transfer to Asparagine residues of
extracellular proteins. (c) Displays the Neisseria gonorrhoeae biosynthesis pathway, proposed for oligosaccharide production and transfer to Serine (and/
or Threonine) residues of extracellular proteins.
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pgl gene cluster. Subsequently, a heptasaccharide is formed
by the consecutive addition of nonmodified, nucleotide-
energized sugars to di-N-acetylbacillosamine by the activity
of the other four glycosyltransferases (PglA, I, H and J). This
heptasaccharide is translocated across the membrane by the
flippase PglK and covalently attached to asparagine residues
by the activity of the oligosaccharyltransferase PglB, which
recognizes a specific acceptor sequence motif in target
proteins (Linton et al., 2005; Kelly et al., 2006). The O-
glycosylation machinery in the Gram-negative human
pathogen Neisseria gonorrhoeae appears to function in the
same way (Stimson et al., 1995) (Fig. 5c). By contrast, in
Eukarya, O-glycans are assembled via sequential coupling of
nucleotide-energized monosaccharides to proteins, and
downstream glycan elaboration (Abu-Qarn et al., 2008). In
N. gonorrhoeae, more than 15 proteins are potentially
glycosylated by this system (Vik et al., 2009). Notably, the
subcellular location predicted for these proteins is in the
periplasm, and they encompass diverse functions involved
in protein folding, disulfide bond formation and respira-
tion. For the majority of the identified proteins, a glycan-
bearing peptide could be identified, and all glycopeptides
contained a serine residue that appears to be essential for
glycosylation. Interestingly, all these Neisseria glycoproteins
share domains with signatures of low complexity that
include their glycosylation sites, while the N-glycosylation
system described for C. jejuni uses a specific acceptor
sequence motif (Vik et al., 2009).
The importance of glycan biology in host–microorganism
communication in the intestine is exemplified by the
observation that Bacteroides species can stimulate the ex-
pression of fucosylated glycoconjugates by the intestinal
epithelia and can subsequently harvest these glycans by the
production of specific degradative enzymes. Moreover,
Bacteroides species have a rare bacterial pathway for the
incorporation of exogenous fucose into their capsular poly-
saccharides and glycoproteins (Comstock & Coyne, 2003).
Recently, eight glycoproteins were identified in Bacteroides
fragilis, which were all predicted to be located in the
periplasm or the outer membrane and involved in protein
folding, protein–protein interactions, peptide degradation
and surface lipoproteins (Fletcher et al., 2009). Three
threonine residues of one of the glycoproteins, embedded
in the conserved Asp-Thr-X motif (where X is a methyl-
containing amino acid), were shown to be involved in
glycoprotein formation. Notably, the Threonine can be
exchanged by a Serine residue without loss of glycosylation
(Fletcher et al., 2009). The same study identified the locus
(lfg) involved in glycosylation in B. fragilis, which encodes
five glycosyltransferases and a flippase. Consistently, muta-
tion of the lfg locus resulted in a loss of protein glycosyla-
tion, and the mutant displayed a significant defect in in vitro
growth and was not able to compete with the wild-type
strain during colonization of gnotobiotic mice (Fletcher
et al., 2009).
The importance of sugar moieties on the surface of
lactobacilli in functional traits such as adhesion (Lebeer
et al., 2009), and gastrointestinal persistence (Denou et al.,
2008) and adaptation (M.L. Marco, M. Wels, W.M. de Vos,
E.E. Vaughan & M. Kleerebezem, unpublished data) has
been suggested in several studies. However, the polysacchar-
ides involved have also been proposed to contribute to
gastrointestinal functionality in a nonspecific manner by
shielding of adhesins (Denou et al., 2008; Lebeer et al.,
2009). In addition, a recent study using lectin-based gly-
coarrays (Hsu et al., 2006) demonstrated significant differ-
ences in sugar decoration on the surface of intact cells of
L. plantarum WCFS1 harvested from either the logarithmic
or the stationary phase of growth (Fig. 6). As glycan
recognition by eukaryal receptors is a well-established
phenomenon, it is tempting to speculate that these observed
differences could (partially) explain the different in vivo
responses of human intestinal mucosa towards L. plantarum
cells harvested in different growth phases (van Baarlen et al.,
2009). However, this and other suggestions (Konstantinov
et al., 2008) that sugar moieties might be covalently attached
to specific extracellular proteins of lactobacilli are at this
stage based on lectin-affinity studies and are, to the best of
our knowledge, not yet supported by biochemical studies
that address the biosynthesis and molecular structure of the
glycans, and the sugar attachment motifs in the potential
target proteins. Nevertheless, many lactobacilli encode the
capacity to produce several nucleotide sugars, including
UDP-glucose, UDP-galactose, UDP-GlcNaC, sialic acid and
dTDP-rhamnose. Moreover, the genome of L. salivarius
encodes a locus (RLSL00992-995) homologous with the
pglFED region of C. jejuni, suggesting that it might be able
to produce the modified monosaccharide di-N-acetylbacil-
losamine (Fig. 5b). Notably, eight of the complete Lactoba-
cillus genomes in the ERGO database harbor at least one
homologue of PglC, indicating their potential capacity to
attach modified monosaccharides to a lipid carrier on the
cytosolic side of the cell membrane. Moreover, capsular
polysaccharide biosynthesis pathways, including several gly-
cosyltransferases and flippases, have been identified in a
variety of lactobacilli, indicating a machinery to synthesize
lipid-linked oligosaccharides that mechanistically resembles
the general glycosylation systems described above (Fig. 5).
However, identification of the dedicated transferases, re-
sponsible for coupling of these oligosaccharides to specific
amino acids in potential target proteins, is less straightfor-
ward, as these oligosaccharyltransferases might display
relatively low homology and typically have rather broad
substrate specificities (Faridmoayer et al., 2007). Overall,
we are only beginning to appreciate the potential impor-
tance of glycans in the functionality of bacteria, and
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220 M. Kleerebezem et al.
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proteo-glycomics remains virtually unexplored in lactobacilli,
even though it may play a key role in host–microorganism
interactions and may eventually explain (part of) the pro-
posed health beneficial effects conferred by these bacteria.
Molecular analysis of host responses tolactobacilli
Postgenomic approaches to unravel host responses to lacto-
bacilli offer novel avenues to unravel host responses to
lactobacilli at the molecular level. The use of gnotobiotic
mouse models has proven its value as a reductionist model
to specifically address the response of intestinal tissues to a
single bacterial species. This approach was pioneered by the
analysis of the transcriptome responses in gnotobiotic mice
following their mono-association with the gut commensal
Bacteroides thetaiotaomicron, which underlined the broad
functional impact and developmental importance of
host–microorganism communication (Hooper et al., 2001).
Subsequent work included co-colonization with B. thetaio-
taomicron and L. casei DN-114 (or Bifidobacterium longum
or Bifidobacterium animalis), showing that cocolonization
impacted on the behavior of B. thetaiotaomicron in situ,
including a shift in the glycan repertoire targeted by the vast
capacity of this commensal bacterium. In addition, cocolo-
nization with Bacteroides and B. longum elicits highly con-
nected TNF-a and IFN-g networks, showing that both
microorganism–microorganism and host–microorganism
interactions shape the intestinal ecosystem (Sonnenburg
et al., 2006). Host responses to microorganism in the
intestine extend beyond local transcriptome responses in
the mucosal tissues, and include significant influences on
local and systemic mammalian biochemistry (Nicholson
et al., 2005). The influences of probiotic interventions on
local and systemic germ-free mouse metabolism have been
addressed by extensive metabolic profiling of different
Fig. 6. Schematic representation of the approach followed in cell-wall glycomics using Lactobacillus plantarum WCFS1. Whole cells harvested from
either the logarithmic or the stationary growth phase were fluorescently labeled using SYTO83 (Step 1) and hybridized to lectin glycol arrays (Qiagen)
(Step 2). Slides were scanned (Step 3) and the glycol-array analysis software was used to convert lectin signals to affinity and binding fingerprints (Step
4), which was subsequently converted to the presence or the absence of specific sugar moieties (Step 5), using a binding cut-off (dotted line in the
fingerprint graph) based on background values.
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221The extracellular biology of lactobacilli
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mouse tissues. These interventions, which used L. paracasei
or L. rhamnosus, induce microbial population changes in the
intestine and altered metabolite profiles in intestinal tissues
(Martin et al., 2007). The Lactobacillus supplementation in
these mice induces remarkably different bile acid/fecal
microbiota correlation networks and significantly affects
many host metabolic pathways, including lipid profiles,
gluconeogenesis and amino acid and methylamine metabo-
lism (Martin et al., 2008a, b). The unprecedented resolution
and explorative strength of these holistic approaches may
unravel unknown mechanisms of interactions that are on-
going in the intestine, and how they influence local and
systemic physiology by affecting a multitude of molecular
parameters. It is unclear whether specific extracellular func-
tions of the lactobacilli play a key role in the measured host
effects, especially because the response analyses using lacto-
bacilli have focused on the impact at the metabolic level,
which may not predominantly involve the extracellular
functions of the lactobacilli. However, specific extracellular
functions may be important for the metabolic impact of
lactobacilli in the intestinal niche, such as the protein
complexes predicted to be involved in extracellular carbohy-
drate recognition and breakdown (Siezen et al., 2006).
Additionally, extracellular functions of the lactobacilli may
(in part) be responsible for the microbiota composition
changes observed in the humanized germ-free mice (Martin
et al., 2007) and may have had more subtle effects on the
host immune system status, which may not be prominently
reflected in the metabolic state of the tissues. Although these
studies present break-through achievements in the area of
host–microorganism interaction at the molecular level, it is
certainly not a trivial task to extrapolate these results to the
human situation, and human responses to dietary lactoba-
cilli will by definition require human intervention studies.
Nevertheless, these may be strengthened through concepts
or mechanistic insights derived from these mouse studies.
Few of the many clinical studies with Lactobacillus inter-
ventions in humans have addressed local molecular re-
sponses in the intestine. One pioneering study in this field
used a duodenal perfusion system in healthy volunteers to
evaluate mucosal transcriptome responses to perfusion with
an L. plantarum WCFS1 suspension, in comparison with a
placebo perfusion (Troost et al., 2008). Both short-term
(1 h) and long-term (6 h) perfusions elicit differential
expression patterns in the human duodenal mucosa.
Short-term exposure to L. plantarum inhibits fatty acid
metabolism and cell cycle progression in the host epithelia,
while long-term perfusions launch responses associated with
increased lipid metabolism, cellular growth and develop-
ment (Troost et al., 2008). Proteome analysis of the biopsies
taken after prolonged perfusion revealed the induction of
microsomal triglyceride transfer protein, which is known to
play a role in lipid transport as well as in immune modula-
tion (Troost et al., 2008). A subsequent double-blind,
placebo-controlled, randomized, cross-over study protocol
was used to determine the small intestinal transcriptional
responses in human volunteers after consumption of a series
of small-sized, bacteria-containing ‘sports drinks.’ These
studies demonstrated that functionally coherent and distinct
transcriptome responses are launched by the human intest-
inal mucosa following the consumption of different Lacto-
bacillus species (L. acidophilus, L. casei and L. rhamnosus),
which appear to be correlated with the previously reported
effects of these lactobacilli on host physiology and health
(P. van Baarlen, F. Troost, van der C. Meer, G. Hooiveld,
M. Boekschoten, & M. Kleerebezem, unpublished data). The
same intervention protocol showed that remarkably distinct
human mucosal transcriptome responses can be detected for
L. plantarum cells harvested from the logarithmic or the
stationary phase of growth. Specifically, stationary-phase-
harvested cells can elicit extensive immune-modulation-
related transcriptional networks centered on the nuclear
factor kB (NF-kB), including both activating and antago-
nizing pathways. These are virtually unaffected by exposure
to L. plantarum harvested from the logarithmic growth
phase. The molecular response patterns surrounding NF-
kB induced by stationary-phase L. plantarum exposure
could be associated with processes such as tolerance and
adjuvanticity, supporting a role of this bacterium in the
modulation of the mucosal immune system (van Baarlen
et al., 2009). The authors speculate on a role of cell-surface-
associated functions in this growth-phase-dependent host-
response modulation, based on molecular studies that
indicate that cell-wall functions are significantly different in
L. plantarum cells obtained from the logarithmic and
stationary phase of growth (van Baarlen et al., 2009). These
differences include the differential recognition of whole cells
by specific lectins, suggesting growth-phase-dependent glycan
decorations of the bacterial cell wall in this species (Fig. 6).
These unique studies in healthy human volunteers exemplify
the highly specific mucosal responses to dietary lactobacilli
(probiotics) and underline the importance of the mode of
production and delivery of these health-promoting cultures.
Concluding remarks
The lactobacilli form a heterogeneous group of bacteria that
display considerable variation both in terms of molecular
characteristics and in terms of their preferred natural
habitats. This variation definitely also includes variation of
bacterial cell-surface properties that are probably very
important for the functioning of these bacteria, especially
in relation to communication with their environment,
including the communication with diet- and host-derived
factors encountered in the gastrointestinal tract. Although
genomics has contributed to our understanding of the
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222 M. Kleerebezem et al.
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extracellular biology of the members of this genus, many of
the proteins that are targeted to the extracellular SCL in
lactobacilli lack a functional annotation, and in many cases,
their sequence analysis does not proceed beyond the detec-
tion of specific domains, illustrating our limited under-
standing of the functional properties that lactobacilli can
confer to their environment. Accurate (and consistent)
predictions of the exoproteomes of lactobacilli on the basis
of their genome sequence, combined with domain detection
and comparative analysis among strains and species, and
(high-throughput) functional analyses will be essential to
enhance our understanding of this subcategory of Lactoba-
cillus functions.
The postgenomics era has strongly stimulated the identi-
fication of candidate effector molecules of lactobacilli that
are proposed to be involved in conferring a health benefit to
the host via interactions with the intestinal system, includ-
ing direct interactions with host epithelial or immune cells.
Notably, many of these candidate effector molecules are
extracellular, including both proteinaceous factors as well as
components of the cell wall itself (Kleerebezem & Vaughan,
2009). However, our understanding of the molecular mode
of action of host–Lactobacillus interactions is still in its
infancy, as is illustrated by the very small number of truly
validated effector molecules identified to date. Moreover,
the suggestion that lactobacilli may produce extracellular
proteins that are decorated with sugar moieties through
glycosylation is highly intriguing, especially with regard to
the predominance of host receptors that recognize glycan
moieties. In view of the complexity of the host-cell signaling
and response-regulation pathways, it does not seem plausi-
ble that single Lactobacillus molecules drive the entire host
response. These effector molecules should be seen within a
‘background’ of molecular properties that includes a poten-
tially crucial role for the basic building blocks of the
Lactobacillus cell wall (Grangette et al., 2005). Expansion of
the candidate effector molecule repertoire and their valida-
tion in vivo will be essential to specify the molecular
mechanisms that underlie the physiological benefits asso-
ciated with the consumption of these bacteria. In addition,
such knowledge would strengthen the concept of the strain
specificity of probiotics and would contribute to the devel-
opment of advanced procedures and criteria for product
quality control and/or selection of novel probiotic strains.
Identification and validation of health-benefit effector mo-
lecules could facilitate research programs that aim to obtain
improved probiotic strains with enhanced health benefits,
either through targeted genetic engineering or through
screening or adaptive evolution under selective conditions
that stimulate an increase in the relevant function.
Molecular research into host–microorganism interactions
has been dominated by animal model studies that allow
stringent control of potentially confounding factors such as
the host genotype, age and diet. Although this type of
research has yielded a wealth of novel insights and may lead
to highly detailed mechanistic insights, it should be realized
that the human population is characterized by a tremendous
variation in these very parameters. Nevertheless, consistent
molecular responses in the mucosa of healthy human
volunteers can be elicited by Lactobacillus consumption,
indicating that despite the individuality of humans, they
share conserved responses to specific bacteria (van Baarlen
et al., 2009). This does not exclude the possibility that the
physiological consequences of these responses may be highly
individual. Nevertheless, these studies illustrate that valida-
tion of (extracellular) effector molecules of lactobacilli can
be performed by advanced molecular response analyses in
relatively small groups of volunteers. Because there is a
considerable degree of variation among different Lactobacil-
lus strains as well as among human volunteers, it seems
essential that effector molecule validation strategies use
isogenic strains and preferably human studies that follow a
cross-over protocol to increase the chance of successfully
elucidating the consequences of the effector molecule muta-
tion on the molecular host response.
Acknowledgements
This work was supported by the BioRange program of the
Netherlands Bioinformatics Centre (NBIC), which is sup-
ported by a BSIK grant through the Netherlands Genomics
Initiative (NGI). In addition, support was also provided by
the National Foundation for Scientific Research (FNRS), the
Foundation for Training in Industrial and Agricultural Re-
search (FRIA) and the Research Department of the ‘Commu-
naute francaise de Belgique’ (Concerted Research Action).
Pascal Hols is research associate of the FNRS. The authors
thank Daniel Teunissen for his help in constructing Fig. 2.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Inventory of Lactobacillus proteins involved in
protein sorting.
Table S2. Inventory of automatically identified extracellular
proteins in complete Lactobacillus genomes, using LocateP
software suite (Zhou et al., 2008).
Table S3. Inventory of the distribution of LaCOGs in the
Lactobacillus exoproteomes, grouped by conserved function
of the LaCOGs.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
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230 M. Kleerebezem et al.