Lactobacilli

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:

[email protected]

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

FEMS Microbiol Rev 34 (2010) 199–230 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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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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200 M. Kleerebezem et al.

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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).


201The extracellular biology of lactobacilli



























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.



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.



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



http://pfam.sanger.ac.uk/



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



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



http://www.cmbi.ru.nl/lab_exoproteome/




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).



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.



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.



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



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.



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



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



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.



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



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



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



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



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.



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



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.



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



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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