RESEARCH ARTICLE

Insight into the core and variant exoproteomes of

Listeria monocytogenes species by comparative

subproteomic analysis

Emilie Dumas1�, Mickael Desvaux1�, Christophe Chambon2 and Michel Hebraud1,2

1 INRA, UR454 Microbiologie, Saint-Genes Champanelle, France2 INRA, Plate-Forme d’Exploration du Metabolisme, Composante Proteomique, Saint-Genes Champanelle, France

Received: September 26, 2008

Revised: February 17, 2009

Accepted: February 26, 2009

While Listeria monocytogenes is responsible for listeriosis, it is also a saprophytic species with

exceptional survival aptitudes. Secreted proteins are one of the main tools used by bacteria to

interact with their environment. In order to take into account the biodiversity of L. mono-cytogenes species, exoproteomic analysis was carried out on 12 representative strains.

Following 2-DE and MALDI-TOF MS, a total of 151 spots were identified and corresponded to

60 non-orthologous proteins. To categorize and analyze these proteomic data, a rational

bioinformatic approach predicting final subcellular localization was carried out. Fifty-two out

of the 60 proteins identified (86.7%) were indeed predicted as localized in the extracellular

milieu (gene ontology (GO): 0005576). Most of them (65.4%) were actually predicted as

secreted via the Sec translocon. Comparative analysis allowed for proteins found in all or only

in a subset of L. monocytogenes strains to be defined. While the core exoproteome included

most proteins related to bacterial virulence, cell wall biogenesis, as well as proteins secreted

by unknown pathways, a slight variation in the protein members of these categories were

observed and constituted the variant exoproteome. This investigation resulted in the first

definition of the core and variant exoproteomes of L. monocytogenes where corollaries on

bacterial physiology are further discussed.

Keywords:

Biodiversity / Bioinformatic analysis / Extracellular proteome / Listeria monocytogenes /

MALDI-TOF MS

1 Introduction

Listeria monocytogenes is one of the major food-borne

pathogenic bacteria and the etiologic agent of listeriosis, a

rare but very serious infection particularly for high-risk

groups, such as elderly, pregnant women, neonates or

immunocompromised individuals, as it can have lethal

consequences [1]. Despite the fact that equivocal data might

result from phenotypic tests over molecular methods when

identifying L. monocytogenes isolates [2], serotype is still

largely used to discriminate different L. monocytogenesstrains. Among the 13 L. monocytogenes serotypes described,

98% of human listeriosis are associated with strains 4b, 1/2b

Abbreviations: ActA, actin assembly; FEA, flagellum export

apparatus; FPE, fimbrilin-protein exporter; GO, gene ontology;

HMM, hidden Markov model; holins, hole-forming; Iap, inva-

sion-associated protein; IMP, integral membrane proteins; Inl,

internalin; LLO, listeriolysin O; MnSOD, manganese-superoxide

dismutase; MurA, muramidase A; Plc, phospholipase C; PSI-

BLAST, position-specific iterated BLAST; SH3, Src homology-3;

SVM, support vector machine; Tat, twin-arginine translocation;

TBP, tributyl phosphine; TMD, transmembrane domain; Wss,

WXG100 secretion system

�These authors contributed equally to this work and share

first authorship.

Correspondence: Dr. Michel Hebraud, Institut National de la

Recherche Agronomique (INRA), Centre de Recherche de Cler-

mont-Ferrand, UR454 Microbiologie, Equipe Qualite et Securite

des Aliments, Site de Theix, F-63122 Saint-Genes Champanelle,

France

E-mail: michel.hebraud@clermont.inra.fr

Fax: 133-4-73-62-45-81

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3136 Proteomics 2009, 9, 3136–3155DOI 10.1002/pmic.200800765

and 1/2a [3]. In other respects, the infectious cycle of

L. monocytogenes is now clearly established as well as the key

virulence factors involved in the major steps of intracellular

parasitism [4]. Briefly, cell-wall localized proteins internalin

A (InlA) and InlB are involved in adhesion to the surface of

the eukaryote cell and penetration into the host cell viaphagocytosis. Then, released listeriolysin O (LLO) and

phospholipases C (PlcA and PlcB) enable escape from the

phagocytic vacuole, whereas membrane-anchored actin

assembly (ActA) is responsible for actin-based motility

allowing for cell-to-cell spread. From this point and as

observed in other pathogenic bacteria [5], it is clearly

apparent that pathogenicity of L. monocytogenes depends

greatly on its ability to secrete virulence factors, which are

displayed on the bacterial cell surface, released into the

extracellular milieu or injected directly into a host cell [6].

As an ubiquitous species, L. monocytogenes is also able to

deal with stressful environmental conditions as it can grow

at high salt concentrations (up to 10%), in a wide range of

pH (from 4.3 to 9.6) and temperatures (from �0.4 to

45.01C), as well as low water availability (Aw down to 0.90)

[7, 8]. Its ability to form biofilms further increases its

adaptative and survival aptitudes in terms of resistance and

persistence [9, 10]. Thus, although at first glance L. mono-cytogenes is generally introduced as a pathogenic agent, it

should be primarily considered as a saprophytic bacterium

well adapted for survival in the environment [11, 12].

Indeed, the natural ecological niche of all L. monocytogenes is

soil and decaying vegetation, whereas the level of virulence

is highly variable from one L. monocytogenes strain to

another as a significant proportion of isolates is hypoviru-

lent and even apathogenic [13, 14]. This facet is sometimes

skipped by some authors but should not be overlooked as it

stresses that L. monocytogenes biodiversity must be taken into

consideration. Moreover, the molecular mechanisms

underlying the switch to a harmful bacterium are far from

being fully understood [12, 15, 16].

While secreted proteins, either displayed on the bacterial

cell surface or released into the extracellular milieu, play

pivotal roles in the colonization process in the environment

and subversion of the human host, the gating systems

involved in secretion of these effector molecules remain

elusive in L. monocytogenes. Following genomic analysis, six

protein secretion systems could be uncovered in L. mono-cytogenes, namely (i) Sec secretion system, (ii) Tat (twin-

arginine translocation) pathway, (iii) FEA (flagellum export

apparatus), (iv) FPE (fimbrilin-protein exporter), (v) holins

(hole-forming), and (vi) Wss (WXG100 secretion system)

also known as ESX-1 (ESAT-6 secretion-1) in Mycobacteriumspp. [6]. Focusing on one sequenced strain, L. monocytogenes1/2a EGD-e, proteomic analyses of extracellular and cell-wall

proteins were performed but unfortunately without taking

the protein secretion systems involved upstream into

account [17–20]. In order to take the biodiversity into

account and get a more objective overview of the exopro-

teome from L. monocytogenes species, i.e. the subset of

proteins present in the extracellular milieu, analysis of this

subproteome was carried out here on 12 representative

L. monocytogenes strains from different serotypes, origins

and virulence levels [21]. Besides, a rational bioinformatic

approach taking into account protein secretion systems,

subcellular targeting and localization motifs was applied to

proteins identified in order to categorize and analyze

proteomic data. This investigation results in the first

definition of the core and variant exoproteomes of

L. monocytogenes species.

2 Materials and methods

2.1 Bacterial strains and culture conditions

The 12 L. monocytogenes strains analyzed in this study were

obtained from the Pasteur Institute (Paris) and are listed in

Table 1. The strains were carefully chosen as previously

described [21] as they either belonged to serotype 1/2a, 1/2b

or 4b. They were isolated from different origins, i.e.epidemic cases, human asymptomatical carriage and envir-

onment. They were also categorized into two virulence levels

based on the mortality of infected chick embryos [22], where

level A corresponds to virulent strains (100% mortality

within 3 days) and level B corresponds to slightly attenuated

strains (80% mortality in more than 3 days).

Bacterial strains were cultured as previously described in

chemically defined medium MCDB202 (CryoBioSystem)

prior to proteomic analysis [21]. They were grown at 371C

with shaking until late exponential phase (OD600 nm 5 0.9)

before harvesting by centrifugation (15 min, 7500g, 41C).

2.2 2-DE of extracellular proteins

First, the supernatants were filtered on 0.2 mm membranes

and 0.2 mM PMSF was added to inhibit proteases activity.

An aliquot of 0.2 mg/mL Na deoxycholate was added to the

solution, which was incubated 30 min on ice. Na deoxy-

cholate supports protein precipitation that was carried out

by the addition of 10% trichloroacetic acid and incubation

overnight at 41C. After centrifugation (20 300� g, 30 min,

41C), the precipitate was washed with ice-cold acetone and

solubilized in IEF buffer (5 M urea, 2 M thiourea, 2%

CHAPS, 10 mM TrisHCl, in 50% trifluoroethanol and traces

of bromophenol blue).

For the IEF, non-linear pH 3–10 IPG strips were

passively rehydrated for 17.5 h in a reswelling tray with

400 mL of IEF buffer containing 0.3% v/v ampholytes 3–10,

2 mM tributyl phosphine (TBP) and 500 mg of proteins.

The proteins were first subjected to IEF for a total of

66450 Vh (7 h at 50 V, 2 h at 200 V, linear gradient until

1000 V in 2 h, 1 h at 1000 V, linear gradient until 8000 V in

5 h and 8000 V till the end). The strips were equilibrated

twice for 15 min in an equilibration solution (50 mM

Proteomics 2009, 9, 3136–3155 3137

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

TrisHCl, pH 8.8; 6 M urea; 2% w/v SDS; 30% v/v glycerol)

containing 2 mM TBP for the first step and 2.5% w/v

iodoacetamide and traces of bromophenol blue for the

second step. The second dimension (SDS-PAGE) was

carried out with 12.5% acrylamide gel in a Multicell Protean

II XL system (Bio-Rad). The gels were stained with

Coomassie blue, scanned by a GS-800 imaging densitometer

(Bio-Rad) and analyzed using Image Master 2D Platinum

software v5.0 (GE Healthcare).

2.3 Identification of proteins by MALDI-TOF MS

Protein spots separated by 2-DE stained with Coomassie

blue were excised. The gel pieces were destained and

submitted to tryptic digestion. First, the spots were washed

with the destaining solution (25 mM ammonium bicarbo-

nate/5% ACN) and twice with ammonium bicarbonate

(25 mM)/50% ACN). They were then dehydrated with 100%

ACN. The dried gels were reswelled in a solution containing

20 mg trypsin and the proteins in the gel were digested at

least 5 h at 371C. The resulting peptides were extracted

with 100% ACN. After 15 min at 371C, 1 mL of each sample

and a 1 mL saturated CHCA matrix were mixed onto the

MALDI-TOF target. Positive ion MALDI mass spectra were

recorded in the reflectron mode of a MALDI-TOF MS

(Voyager DE-Pro, Perseptive BioSystems) using voyager

software for data collection and analysis. The MS was cali-

brated with a standard peptide solution (Proteomix, LaserBio

Labs). Internal calibration of samples was achieved using

trypsin autolysis peptides. Monoisotopic peptide masses were

used for NCBI non-redundant database searches (http://

www.ncbi.nlm.nih.gov/BLAST/blast_databases.html) with the

MASCOT v.2.1 software, or Profound (http://www.unb.

br/cbsp/paginiciais/profound.htm) when MASCOT failed to

assign. The maximum fragment ion mass tolerance was set up

at 725 or 50 ppm and possible modification of cysteines by

carbamidomethylation as well as oxidation of methionine

could be considered.

2.4 Bioinformatic analyses

Bioinformatic analyses were performed from web-based

servers or under a Unix environment and Sun Grid Engine

from a Topaze server homed at MIG (Mathematiques

Informatique et Genomes) Research Unit (INRA).

N-terminal signal peptides were predicted combining

results [23] from (i) SPScan, an implementation of von

Heijne’s weight matrix approach with McGeoch criteria where

prompted parameters and optional parameter ADJustscores

were used [24, 25], (ii) SignalP v2.0 and v3.0 using both a

neural network and hidden Markov model (HMM) [26],

(iii) Phobius based on a HMM combining transmembrane

a-helices topology and signal peptide prediction [27], (iv)

SOSUIsignal based on tripartite signal peptide structureTab

le1.

Str

ain

so

fL.

mo

no

cyto

gen

es

use

din

this

stu

dy

Co

de

Str

ain

Sero

typ

eO

rig

inV

iru

len

ceR

efe

ren

ces

AC

LIP

80459

4b

Ep

idem

icA

[21,

104]

BC

LIP

90602

1/2

bE

pid

em

icA

[21]

CC

LIP

92347

1/2

aE

pid

em

icA

Past

eu

rIn

stit

ute

Co

llect

ion

DE

GD

-e1/2

aE

pid

em

icA

[78,

105]

EC

LIP

93667

4b

Carr

iag

eA

[106,

107]

FC

LIP

93679

4b

Carr

iag

eA

[106,

107]

GC

LIP

93672

1/2

bC

arr

iag

eB

[106,

107]

HC

LIP

93677

1/2

aC

arr

iag

eB

[106,

107]

IC

LIP

93665

4b

En

vir

on

men

tA

[21]

JC

LIP

93649

1/2

aE

nvir

on

men

tA

[21]

KC

LIP

93663

1/2

aE

nvir

on

men

tB

Past

eu

rIn

stit

ute

Co

llect

ion

LC

LIP

93657

1/2

aE

nvir

on

men

tB

Past

eu

rIn

stit

ute

Co

llect

ion

Th

e12

L.

mo

no

cyto

gen

es

stra

ins,

cod

ed

fro

mA

toL

for

the

pu

rpo

ses

of

this

invest

igati

on

,(i

)exh

ibit

ed

dif

fere

nt

sero

typ

es

(1/2

a,

1/2

bo

r4b

),(i

i)w

ere

iso

late

dfr

om

div

ers

eo

rig

ins

(ep

idem

icca

ses,

asy

mp

tom

ati

cal

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es

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den

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on

men

t),

(iii

)w

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rize

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od

iffe

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ule

nce

levels

(Aan

dB

)b

ase

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bry

os

(100

an

d80%

mo

rtali

tyw

ith

in3

days,

resp

ect

ively

).

3138 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

analysis [28], (v) PSORTb a support vector machine (SVM)

[29, 30] (vi) PrediSi [31] and (vii) Signal-3L [32]. These tools

were trained on prokaryotes or Gram-positive bacteria when-

ever possible with truncation disabled. Tat signal peptide

prediction was performed from TatP v1.0 [33] and TATFIND

v1.4 [34]. Pseudopilin-like signal peptides (class 3) were sear-

ched with ScanProsite syntax [35] for consensus motif

[AG]F[TS]LX[EF] located between the N- and H-domains as

recently described in Listeria [6]. Prediction of non-classical

secreted proteins, i.e. lacking a signal peptide, was performed

from SecretomeP v2.0 trained on bacteria [36].

For the identification of lipoproteins, sequences were

submitted to DOLOP [37], LipoP v1.0 [38], SPEPLip [39],

LipPred [40] and scanned by ScanProsite [35] with both

PS51257 profile and G1LPP pattern, i.e. [MV]-X(0,13)-[RK]-

DERKQ(6,20)-[LIVMFESTAG]-[LVIAM]-[IVMSTAFG]-[AG]-

C [41].

Modular architecture of proteins was analyzed from COG

v1.0 [42] using reverse position-specific BLAST [43] and

using a HMM [44] from InterPro v4.3 [45], Pfam v21.0 [46],

SMART v5.1 [47], TIGRfam v6.0 [48], SCOP v1.71 [49, 50],

PIRSF [51] and Prosite v20.7 [52] databases. WXL

domains were found scanning for the motif [LI][TE]W[TS]L

with ScanProsite where the C-terminal location of the

motif was also taken into account [53]. When needed,

e.g. for no significant or inconclusive matches, position-

specific iterated BLAST (PSI-BLAST) [43] searches

were executed against UniProt until convergence [54].

All searches were performed with E-value cutoff set

at 10�3.

Transmembrane a-helices were predicted combining (i)

HMMTOP v2.0 [55], (ii) TMHMM v2.0 [56], (iii) SOSUI v1.1

[57], (iv) THUMBUP v1.0 [58], (v) MEMSAT v3.4 [59] and

(vi) UMDHMMTMHP v1.0 [58].

These analyses were completed with results from SVMs

predicting the subcellular localization of proteins, i.e.SubLoc v1.0 [60] and LocTree [61] trained on prokaryotes,

CELLO v2.5 [62] and PSORTb v2.0.4 trained on Gram-

positive bacteria [30].

3 Results and discussion

The diversity of the extracellular proteomes of L. mono-cytogenes species was investigated analyzing a panel of 12

representative strains carefully chosen as previously descri-

bed (Table 1) [21]. In order to separate the proteins over a

wide pI range, a non-linear pH 3–10 IPG strip was used for

IEF separation in the first dimension. To facilitate

comparisons, a reference 2-DE gel was realized from a

mixture of an equivalent quantity of proteins from the 12

L. monocytogenes strains (Fig. 1). Two clusters of proteins

could be distinguished as the majority of the protein spots

were localized in a region between pH 4.0 and 6.0, whereas

some very abundant spots were present in the basic region.

Protein spots were sampled from gels corresponding to each

L. monocytogenes species and further identified from their

peptide mass fingerprinting obtained by MALDI-TOF MS

and MASCOT or Profound interrogation against the

Firmicutes database (NCBInr). The gels allowed for a

total of 151 spots to be resolved and identified. Several

proteins were resolved as two or more spots on the 2-DE,

which could be caused by post-translational modifications.

As listed in Table 2 and after manual inspections of the

output to remove protein fragments, redundant proteins (i.e.amino acid sequences 100% identical) as well as protein

orthologues, these 151 protein spots corresponded to 60

non-orthologous proteins (see also Supporting Information

Table S1).

3.1 Predicted subcellular localization of identified

proteins

In order to categorize the 60 proteins extracted from culture

supernatants of these 12 L. monocytogenes strains and iden-

tified by MALDI-TOF MS, bioinformatic analyses were

carried out. The rationale behind this approach is presented

in Fig. 2, and the summary of predictions is given as

Supporting Information Table S1.

3.1.1 Signal peptide and protein secretion system

prediction

Firstly, protein sequences were scanned for the presence of

N-terminal signal peptides using a large variety of bioin-

formatic tools, namely SPScan, SignalP, Phobius, SOSUI-

signal, PSORTb and PrediSi as described in Section 2,

which led to ten individual predictions. Fourty-six proteins

were predicted as possessing a signal peptide by at least one

tool, including ten proteins predicted as such by all predic-

tors. If at least half of the prediction tools gave a significant

positive result, a protein was predicted as possessing a

signal peptide, i.e. 37 proteins. When less than five out of

ten predictors identified a signal peptide, i.e. nine proteins,

proteins were further checked for subcellular localization

and function using appropriate prediction tools, namely

PSORTb, SubLoc, SecretomeP, CELLO and LocTree.

Finally, 42 proteins were predicted as possessing an N-

terminal signal peptide.

While a protein bearing an N-terminal signal peptide is

targeted to the membrane, it is not systematically translo-

cated via the Sec translocon. Further in silico analyses were

carried out in order to find and discriminate different types

of signal peptide. TatP and TATFIND searches failed to

identify Tat signal peptide and thus potential protein

translocated by this secretion system. Similarly, ScanProsite

searches for class 3 signal peptide motifs as recently

described in Listeria [6] could not identify Type 4 prepilins

potentially translocated by the FPE. The remaining proteins

lacking a signal peptide were checked as substrates for

Proteomics 2009, 9, 3136–3155 3139

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

alternative secretion systems. Using a database generated

from recent reviews on protein secretion in Listeria [6], PSI-

BLAST searches failed to identify protein substrates of FEA,

holins and Wss. However, following a SecretomeP search

and literature survey, 17 additional proteins were predicted

as secreted by unknown secretion systems. Finally, 42

proteins identified with an N-terminal signal peptide were

predicted as translocated via the Sec translocon and 17

proteins lacking a signal sequence were predicted as secre-

ted via unknown secretion systems.

3.1.2 Prediction of cell-envelope-binding motifs

The presence of N-terminal signal peptides (i.e. containing at

least N- and H-domains) unmistakably indicates proteins are

targeted to the cytoplasmic membrane but, contrary to what is

sometimes wrongly assumed in the literature, it is no guarantee

that these proteins are secreted into the extracellular milieu.

Indeed, such a protein can either (i) remain associated to the

cytoplasmic membrane, (ii) remain associated to the cell wall or

(iii) be released into the extracellular milieu.

3.00 4.50 4.95 5.95 6.50 7.00 7.80 8.35 9.65 10.00

64

32

16

8

pH

MM(kDa)

Figure 1. Reference 2-DE gel composed from a mixture of an equivalent quantity of extracellular proteins from each of the 12

L. monocytogenes strains investigated. The labels appear in white for the core proteome and in gray for the variant proteome. The

corresponding spot numbers indicated in Tables 2 and 3 are shown in Supporting Information Fig. 1S.

3140 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3.1.2.1 Lipoproteins

Therefore, proteins predicted as Sec-dependent were further

scanned for the presence of class 2 signal peptides, i.e.presence of lipobox, which invariably includes a cysteine

residue located just after the signal peptide C-domain.

Following DOLOP, LipoP, SPEPLip, LipPred searches as

well as ScanProsite using both PS51257 profile and G1LPP

pattern [41], a protein was identified as lipoprotein if at least

three out of six individual predictions gave a significant

positive result, i.e. five proteins. Captivatingly, all the lipo-

proteins identified here in the extracellular milieu display a

glycine residue at position 12 of the predicted Type II signal

peptidase cleavage site. Indeed, this amino acid at this

particular position is considered as of major importance in

the lipoprotein release into the supernatant in Gram-posi-

tive bacteria [63–65]. Although lipoproteins are primarily

considered as anchored to the cytoplasmic membrane, some

of them can to some extent be released extracellularly.

3.1.2.2 LPXTG-like proteins

Motif searches were further carried out from InterPro,

Pfam, Smart, TIRGfam, SuperFamily, PIRsf and Prosite in

order to identify cell-wall retention motifs [23]. Two proteins

were predicted with a C-terminal LPXTG domain

(IPR001899, PF00746, TIGR01167; 2.4� 10�7rE-values

r9.7� 10�4) and thus would be covalently anchored to the

bacterial cell wall by sortases [66]. No YSIRK motif

(IPR005877, PF04650, TIGR01168) could be identified

within Sec-dependent signal peptides of these proteins;

when present, this motif is systematically associated

with an LPXTG motif and is required for efficient protein

secretion [67].

3.1.2.3 LysM proteins

Three proteins were predicted with LysM domains found in

two or four copies (IPR002482, PF01476, SM00257,

SSF54106; 3.7� 10�21rE-valuesr2.0� 10�12). LysM

28

Cell wall(GO:0005618)

12

FPE0

Yes

No

Tat0

Holin0

FEA0

Wss0

Unknown17

No

Non-orthologous proteins60

NoYes

Sec42

1

TMD?

IMP9

Cytoplasm(GO:0005737)

15

Extracellular(GO:0005576)

51

LPXTG2

LysM3

GW3

Signal peptide?

Secretion system? Yes

Cell envelope binding motif?

Lipobox5

seYoN

8124

Muramidase3

Cell wall degradation?

Cell wallamidase

2

5

Yes

NlpC/P602

Final localization? Membrane(GO:0005886)

13

3D1

3 141 420

2 12

1

11

Yes

Yes

25

oNoN No NoNo Yes oNoN Yes No Yes No

No No

No oNoN No

120 988 2 14414

8

3

ChW1

No

No

11

WXL1

No

1

No

1

5

5

28

Cell wall(GO:0005618)

12

FPE0

Yes

No

Tat0

Holin0

FEA0

Wss0

Unknown17

No

Non-orthologous proteins60

NoYes

Sec42

1

TMD?

IMP9

Cytoplasm(GO:0005737)

15

Extracellular(GO:0005576)

51

LPXTG2

LysM3

GW3

Signal peptide?

Secretion system? Yes

Cell envelope binding motif?

Lipobox5

seYoN

8124

Muramidase3

Cell wall degradation?

Cell wallamidase

2

5

Yes

NlpC/P602

Final localization? Membrane(GO:0005886)

13

3D1

3 141 420

2 12

1

11

Yes

Yes

25

oNoN No NoNo Yes oNoN Yes No Yes No

No No

No oNoN No

120 988 2 14414 3

ChW1

No

No

11

WXL1

No

1

No

1

5

5

8

Figure 2. Schematic representation of the bioinformatic analyses carried out to categorize the 60 non-othologous proteins identified in the

extracellular milieu of L. monocytogenes strains. In order to predict the final subcellular localization of the proteins identified following a

proteomic approach, the rationale of the in silico analysis carried out can be summarized as answering to six successive and fundamental

questions related to (i) the presence of N-terminal signal peptides, (ii) the secretion systems used, the presence of (iii) cell-envelope-

binding motifs, (iv) cell-wall degradation motifs or (v) transmembrane domains, and (vi) their assignment to subcellular compartments

recognized in Gram-positive bacteria. Detailed results are given as Supporting Information Table S1.

Proteomics 2009, 9, 3136–3155 3141

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

domains bind directly to peptidoglycan and binding is

hindered by secondary cell-wall polymers, supposedly lipo-

teichoic acids [68]. One of them, i.e. the invasion associated

protein P60 (P21171), also exhibits homology with an Src

homology-3 (SH3) domain (IPR003646, SSF50044, SM00287;

4.1� 10�15rE-valuesr1.3� 10�14), which belongs to the

SH3 clan (CL0010); it further appeared that these domains

were more specifically bacterial SH3 of Type 3 (IPR013247,

PF08239; 3.1� 10�20rE-valuesr2.1� 10�19).

3.1.2.4 Proteins

Three proteins were predicted with GW modules with

copies ranging between one and four (SSF82057;

3.0� 10�23rE-valuesr1.7� 10�12). This motif allows for

non-covalent interaction with lipoteichoic acids, where the

higher the number of GW modules, the stronger the

attachment [69]. However, proteins exhibiting only one GW

module are essentially present in the extracellular milieu

and not found attached to the bacterial cell surface [69, 70].

Therefore, two out of three proteins with GW modules were

primarily predicted as cell-wall-associated at this stage of the

analysis, as namely proteins of unknown function Q8Y572

and Q71WI7 bore two and four GW modules, respectively,

whereas autolysin Q4EHT4 exhibited only one GW module.

3.1.2.5 Other proteins with cell-wall-binding motifs

A serine protease (Q71YE5) bears three copies of a

ChW motif (IPR006637, SM00728, PF07538; 2.5� 10�16

rE-valuesr5.6� 10�11). As GW proteins, ChW proteins

contain a highly conserved Gly-Trp dipeptide motif origin-

ally described in Clostridium acetobutylicum where it was

suggested to enable protein cell surface anchoring [71] and

to be involved in a novel extracellular macromolecular

system. Similarly, protein CscB (Q8Y9E5) was suggested as

participating in cell-surface complexes for plant carbohy-

drate utilization [72]. Indeed, CscB exhibits a newly uncov-

ered domain WXL-mediating cell-wall attachment [73]. All

proteins bearing cell-wall-binding domain(s) were predicted

as secreted in a Sec-dependent manner.

3.1.3 Prediction of motifs involved in cell-wall

degradation

In addition to the autolysin exhibiting one GW module

(Q4EHT4), a protein originally annotated as hypothetical

(Q8Y707) first appeared clearly expressed and would also be

an N-acetylmuramoyl-L-alanine amidase (COG0860;

E-values 5 1.0� 10�48; IPR002508, PF01520; E-values 5 3.6

� 10�64), which further exhibits three SH3 domains of Type 3

(2.1� 10�24rE-valuesr1.8� 10�8). In addition, three

muramidases involved in the flagellar assembly were uncov-

ered (COG1705; 3.0� 10�52rE-valuesr9.0� 10�41) [74]; two

of them were GW proteins and one of them a LysM protein.

One protein (Q71WQ6) exhibited a 3-D domain (IPR010611,

PF06725; E-values 5 1.5� 10�35), which is a peptidase-like

active site containing three conserved aspartate residues and

involved in peptidoglycan turnover. This enzyme was also

predicted as a LysM protein. Including invasion associated

protein (Iap), also called P60 (protein of 60 kDa) or cell wall

hydrolase A, two proteins exhibited an NlpC/P60 domain

(IPR000064, PF00877; 3.5� 10�53rE-valuesr6.2� 10�51)

characteristic of cell-wall peptidases [75]; one of them (P21171)

has LysM domains. As active sites of all these cell-wall

degradation enzymes have affinity for cell-wall components,

they are most likely present within this subcellar compart-

ment. Contrary to what is sometimes assumed though, the

presence of such domains should not be considered as cell-

wall-binding motifs per se since the primary function of these

enzymes is to cleave cell-wall components according to

whether they find a new cleavage site or are released into the

extracellular milieu. Such proteins were then predicted as

localized in two final subcellular compartments, i.e. cell wall

and extracellular milieu. All cell-wall degradation enzymes

were primarily predicted as secreted via Sec.

3.1.4 Prediction of transmembrane a-helices

All proteins were further analyzed for the presence of trans-

membrane a-helices using HMMTOP, TMHMM, SOSUI,

THUMBUP, UMDHMMTMHP and MEMSAT. A protein

was predicted as possessing a transmembrane domain (TMD)

if at least three out of six tools gave a significant positive result.

In addition, the presence of motifs (e.g. LPXTG), signal

peptides and positions of TMD were carefully taken into

consideration for the prediction of integral membrane proteins

(IMP). In the first instance, 48 proteins were predicted as with

transmembrane a-helices but excluding signal peptide

H-domain and LPXTG region, IMPs were reestimated down

to nine. Only three of them did not display Sec-dependent

N-terminal signal peptides. Five proteins displayed only one

putative a-helical TMD and the remaining ones were predicted

as possessing at least two transmembrane a-helices, and up to

five in one of them, i.e. a sulphatase (Q8Y8H6). Excluding

their class 2 signal peptide H-domain, one out of the six

predicted lipoproteins exhibited two TMDs, i.e. AA3-600 quinol

oxidase subunit II (QoxA; Q8YAV0), and would thus be IMP

covalently attached to membrane long chain fatty acids.

However, shedding of QoxA into the culture supernatant was

previously reported in other Gram-positive bacteria as this

lipoprotein exhibits a Gly residue at position 12 [76].

3.1.5 Final prediction of subcellular localizations

All remaining proteins were further submitted to four SVMs

dedicated to bacterial subcellular localization. Four

localizations were considered, i.e. cytoplasmic, membrane,

cell wall and extracellular. Protein was predicted in a given

subcellular compartment if at least two out of four tools

predicted it. These results were combined to previous

3142 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

analyses for the final prediction of subcellular localization.

Following recommendations from the gene ontology (GO)

Consortium to unambiguously define protein localization in

a Gram-positive bacterium [77], GO numbers were given, i.e.cytoplasm (GO: 0005737), cytoplasmic membrane (GO:

0005886), cell wall (GO: 0005618) and extracellular milieu

(GO: 0005576). Finally, 15 proteins were predicted as cyto-

plasmic with 14 also predicted as extracellular. Thirteen

proteins were predicted as membrane associated and more

exactly as intrinsic to membrane (GO: 0031226) including

nine IMPs, i.e. integral to membrane (GO: 0005887), and

four lipoproteins without TMDs, i.e. anchored to membrane

(GO: 0046658). Twelve proteins were predicted as cell-wall

localized. Among the 51 proteins predicted as extracellular,

20 were predicted as secreted via the Sec pathway and finally

released into the extracellular milieu. Besides extracellular

localization, 31 out of these 51 proteins were predicted with

multiple final subcellular localization either as also (i)

present in the cell wall, i.e. nine proteins, (ii) localized at the

membrane, i.e. eight proteins or (iii) primarily cytoplasmic,

i.e. 14 proteins (Fig. 1 and Table 2). Altogether, 51 out of 60

non-orthologous proteins identified here (85%) could clearly

be predicted as extracellular.

3.2 Core and variant exoproteomes of

L. monocytogenes species

Comparison of exoproteomes of the 12 L. monocytogenesstrains investigated here revealed that 43 out of the 60 non-

orthologous proteins identified were commonly found in the

extracellular milieu of all strains (Tables 2 and 3, Fig. 2 and

Supporting Information Fig. 1S). As already mentioned,

several proteins were resolved as more than one single spot

that could result from post-translational modifications and/

or orthologues with different Mr and/or pI. Even though

some of these proteins were expressed in the 12 L. mono-cytogenes strains, some protein spots were specifically

present only in a subset of strains suggesting strain-specific

post-translational modifications (Table 2 and Fig. 2). Besides

this core exoproteome, 17 proteins appeared as expressed

only in some L. monocytogenes strains as these proteins

were at the most only present in or absent from one strain

(Table 3 and Fig. 2).

As expected from the high proportion of genes annotated

as hypothetical in L. monocytogenes genome in GenBank [78],

25 out of the 60 proteins identified here had unknown

functions (Supporting Information Table S1). In addition,

considering that automatic annotations may give false func-

tional identification [79], all the annotations were firstly

verified seeking for matches against various databases,

namely Pfam, Prosite, SMART, TIGRfam, SCOP and PIRSF.

For proteins resulting in inconclusive or no matches, PSI-

BLAST searches were performed against UniProt until

convergence was reached. Besides confirming and improving

original annotations, this approach allowed for the number of

proteins with unpredicted functions to be reduced to seven

(Tables 2 and 3 and Supporting Information Table S1).

3.2.1 Virulence factors

Several virulence factors were identified in the culture super-

natant of the different L. monocytogenes strains investigated. Key

extracellularly secreted virulence factors LLO (Spots 1038, 1041,

1418, 1461, 3743 and 3744) and phosphatidylcholine Plc (PlcB;

Spots 2082, 2085, 2088, 2174, 2177, 2178, 2208, 2267, 2280 and

2290) were identified in all the L. monocytogenes strains investi-

gated, though phosphatidylinositol Plc (PlcA; Spot 1957) was

absent from L. monocytogenes CLIP90602 (Tables 2 and 3 and

Supporting Information Fig. 1S). While LLO is required for

L. monocytogenes escape from vacuoles formed after inter-

nalization and cell-to-cell spreading, the combined action of

PlcA and PlcB allows for efficient hydrolysis of vacuolar

membranes [80]. ActA mediates the polarized actin tail forma-

tion within the host cell that propels the bacteria toward the

plasmic membrane, thus enabling cell-to-cell spreading [81].

ActA is a Type I integrated membrane protein but its limited

release from the bacterial cell surface was previously reported

[82]. Similarly, a membrane-anchored immunogenic protein

that elicits pathogen-specific CD41 T-cell responses was also

found in the culture supernatant (Table 2 and Supporting

Information Fig. 1S; Spots 1653, 1799 and 2295). Although its

role in pathogenesis remains unclear [83], the presumably Sec-

secreted InlC (Table 2 and Supporting Information Fig. 1S;

Spots 2076, 2079, 2102, 2155, 2184, 2181 and 2191), which

belongs to the third and last subfamily of Inl [84], was as

expected found extracellularly in the 12 L. monocytogenes strains

investigated. Antioxidant potential of manganese-superoxide

dismutase (MnSOD; Table 2 and Supporting Information

Fig. 1S; Spots 2441, 2472, 2488 and 2491) is critical in

L. monocytogenes pathogenesis [85]. Despite this protein lacking

an N-terminal signal peptide, MnSOD is secreted in a route

involving the alternative cytosolic ATPase SecA2, a paralogue of

SecA, that most certainly converges to the protein conducting-

channel constituted by the Sec translocon in L. monocytogenes[6, 85]. Surprisingly enough, MnSOD was neither predicted nor

identified as part of the exoproteome in previous L. mono-cytogenes proteomic analysis by Trost et al. [19]. Along with

MnSOD, Iap (Table 2 and Supporting Information Fig. 1S;

Spots 1085, 2708 3746 and 3747) is found in the supernatant of

the 12 L. monocytogenes strains and is secreted in a SecA2-

dependent manner [86].

3.2.2 Enzymes of cell-wall degradation/maturation

A number of proteins primarily involved in cell-wall degra-

dation and/or maturation were present extracellularly as

they are most certainly released from the cell wall as a result

of their catalytic activity and/or cell wall turnover, which

could further explain the presence of proteins primarily

Proteomics 2009, 9, 3136–3155 3143

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le2.

Co

reexo

pro

teo

me

of

L.

mo

no

cyto

gen

es

speci

es

foll

ow

ing

pro

tein

iden

tifi

cati

on

by

MA

LD

I-T

OF

MS

Pro

tein

desc

rip

tio

nU

niP

rotK

BID

a)

Pre

dic

ted

sub

cell

ula

rlo

cali

zati

on

b)

Sp

ot

E-v

alu

ec)

MM

(kD

a)d

)p

IP

ep

tid

em

atc

hin

gS

eq

uen

ceco

vera

ge

e)

L.

mo

no

cyto

gen

es

stra

inf)

Th

eo

Exp

Th

eo

Exp

Pre

sen

ceA

bse

nce

Vir

ule

nce

dete

rmin

an

ts

Lis

teri

oly

sin

O,

Hly

P13128

EC

1038

1.9�

10�

15

58.7

57.4

7.6

6.8

23

49

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1041

9.4�

10�

18

58.7

57.4

7.6

6.6

22

45

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1461

9.6�

10�

558.7

46.7

7.6

7.6

14

32

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1418

8.0�

10�

558.7

48.1

7.6

6.8

11

29

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

3743

1.9�

10�

10

58.7

60.7

7.6

6.9

18

40

A,C

,D,E

,F,H

,I,J

,K,L

B,G

3744

1.2�

10�

13

58.7

60.7

7.6

7.2

19

39

A,C

,D,E

,F,H

,I,J

,K,L

B,G

Ph

osp

hati

dylc

ho

lin

ep

ho

sph

oli

pase

C,

Plc

BP

33378

EC

2088

3.7�

10�

12

33.2

27.0

8.4

6.9

15

50

A,C

,D,E

,F,G

,H,I,J

,K,L

B2177

3.0�

10�

633.2

24.7

8.4

7.0

14

39

A,C

,D,E

,G,H

,I,J

,K,L

F,B

2178

5.4�

10�

533.2

24.6

8.4

7.0

10

39

A,C

,D,E

,G,H

,I,J

,K,L

F,B

2174

1.7�

10�

233.2

24.7

8.4

7.7

936

C,D

,E,F

,G,H

,I,J

,K,L

A,B

2280

5.3�

10�

433.2

22.6

8.4

7.4

13

41

B,C

,D,E

,F,H

,I,J

,K,L

A,G

2082

9.1�

10�

433.2

27.1

8.4

7.0

526

C,D

,E,F

,H,I

,J,K

,LA

,B,G

2085

3.7�

10�

833.2

27.0

8.4

7.0

13

32

C,D

,F,H

,J,K

,LA

,B,E

,G,I

2267

2.0�

10�

333.2

22.6

8.4

7.8

12

34

C,D

,F,H

,J,K

,LA

,B,E

,G,I

2290

1.9�

10�

333.2

22.6

8.4

7.0

13

43

A,B

,C,G

,H,L

D,E

,F,I

,J,K

2208

4.3�

10�

333.2

23.2

8.4

7.0

11

29

A,D

,E,F

,G,L

B,C

,H,I

,J,K

Act

in-a

ssem

bly

-in

du

cin

gp

rote

in,

Act

AP

33379

MB

1031

1.7�

10�

4�

70.3

59.3

5.0

4.6

14

30

A,B

,D,E

,H,L

C,F

,G,I

,J,K

673

6.1�

10�

12�

70.3

59.2

5.0

4.7

15

36

A,B

,D,E

,F,G

,H,I,J

,K,L

C609

2.2�

10�

2�

70.3

74.0

5.0

4.8

614

A,B

,D,E

,F,G

,H,I,J

,K,L

C757

5.8�

10�

5�

70.3

69.0

5.0

4.8

10

23

A,B

,D,E

,F,G

,H,I,J

,K,L

C1159

6.0�

10�

270.3

55.8

5.0

4.9

10

17

A,B

,E,F

,G,I

,J,L

C,D

,H,K

979

2.3�

10�

470.3

60.2

5.0

4.4

11

18

A,B

,D,E

,G,H

,I,J

,LC

,F,K

1099

6.3�

10�

470.3

57.5

5.0

4.6

10

18

A,B

,E,G

,H,I,J

,LC

,D,F

,K756

4.2�

10�

870.3

69.1

5.0

4.8

18

34

A,B

,D,E

,F,G

,H,I,J

,K,L

C942

1.6�

10�

270.3

62.0

5.0

4.6

10

18

A,B

,E,G

,H,I,J

,LC

,D,F

,K612

1.2�

10�

11

70.3

70.3

5.0

4.7

17

33

A,C

,D,E

,F,G

,H,I,J

,K,L

B1137

6.6�

10�

470.3

56.2

5.0

4.5

10

18

A,B

,E,G

,H,J

,LC

,D,F

,I,K

CD

41

T-c

ell

-sti

mu

lati

ng

an

tig

en

,T

csA

Q48754

MB

1653

3.1�

10�

638.4

35.8

5.0

4.6

12

35

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2295

3.6�

10�

338.4

22.6

5.0

5.0

825

LA

,B,C

,D,E

,F,G

,H,I

,J,K

1799

1.2�

10�

438.4

34.3

5.0

4.6

933

LA

,B,C

,D,E

,F,G

,H,I

,J,K

Inte

rnali

nC

,In

lCQ

8Y

6A

8E

C2076

1.9�

10�

333.0

28.2

6.1

5.1

10

38

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2155

5.6�

10�

333.0

25.1

6.1

4.9

832

A,B

,C,D

,E,F

,G,H

,I,J

,KL

2191

1.5�

10�

11

33.0

24.2

6.1

6.1

13

44

A,B

,C,D

,F,G

,H,J

,K,L

E,I

2184

1.1�

10�

233.0

24.4

6.1

5.6

618

A,C

,D,F

,G,H

,J,K

,LB

,E,I

2181

3.8�

10�

11

33.0

24.5

6.1

5.0

15

50

C,D

,F,G

,H,J

,KA

,B,E

,I,L

2102

1.9�

10�

233.0

28.0

6.1

5.0

830

C,D

,H,K

A,B

,E,F

,G,I,J

,L2079

8.9�

10�

633.0

24.5

6.1

5.6

13

46

AB

,C,D

,E,F

,G,H

,I,J

,K,L

3144 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le2.

Co

nti

nu

ed

Pro

tein

desc

rip

tio

nU

niP

rotK

BID

a)

Pre

dic

ted

sub

cell

ula

rlo

cali

zati

on

b)

Sp

ot

E-v

alu

ec)

MM

(kD

a)d

)p

IP

ep

tid

em

atc

hin

gS

eq

uen

ceco

vera

ge

e)

L.

mo

no

cyto

gen

es

stra

inf)

Th

eo

Exp

Th

eo

Exp

Pre

sen

ceA

bse

nce

Man

gan

ese

-su

pero

xid

ed

ism

uta

se,

Mn

SO

DP

28764

EC

,C

P2472

2.5�

10�

11

22.6

22.6

5.2

5.0

13

70

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2491

8.9�

10�

622.6

22.6

5.2

4.8

744

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2488

1.4�

10�

422.6

22.6

5.2

5.0

947

E,F

,G,I

,JA

,B,C

,D,H

,K,L

2441

3.9�

10�

11

22.6

22.6

5.2

5.0

13

92

A,E

,F,G

,I,J

,K,L

B,C

,D,H

Invasi

on

-ass

oci

ate

dp

rote

in,

Iap

P21171

EC

,C

W3746

5.8�

10�

250.3

35.3

9.3

9.2

10

28

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

3747

8.2�

10�

450.3

34.8

9.3

9.1

11

27

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1085

4.7�

10�

950.3

53.0

9.3

9.3

14

38

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2708

1.6�

10�

250.3

18.0

9.3

6.4

846

A,B

,E,F

,G,I

A,B

,E,F

,G,I

Cell

wall

deg

rad

ati

on

an

db

iog

en

esis

P45

pep

tid

og

lyca

nly

tic

pro

tein

,N

LP

/P60

fam

ily,

Sp

lQ

9R

E04

EC

,C

W1485

2.4�

10�

13

42.7

44.0

8.6

7.2

15

38

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1489

4.8�

10�

13

42.7

44.0

8.6

7.8

15

38

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1484

5.6�

10�

642.7

44.0

8.6

6.7

13

34

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1505

4.9�

10�

442.7

45.0

8.6

6.2

724

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

N-a

cety

lmu

ram

oyl-

L-a

lan

ine

am

idase

Q8Y

707

EC

,C

W1436

3.5�

10�

12

46.0

47.4

5.5

5.0

17

49

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1435

1.5�

10�

13

46.0

47.4

5.5

4.9

16

47

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1455

9.5�

10�

11

46.0

47.4

5.5

4.8

14

44

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2103

2.4�

10�

14

46.0

27.4

5.5

5.4

17

49

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1444

1.1�

10�

14

46.0

47.4

5.5

5.2

19

53

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1428

6.9�

10�

546.0

47.6

5.5

5.4

721

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1438

8.4�

10�

546.0

47.6

5.5

5.4

11

26

C,E

,F,H

,I,K

,LA

,B,D

,G,J

Au

toly

sin

,M

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Q8Y

3Y

8E

C,

CW

1601

1.2�

10�

3�

63.5

38.0

9.7

6.1

68

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Mu

ram

idase

flag

ell

um

-sp

eci

fic

Q71W

I7E

C,

CW

1122

2.0�

10�

11

57.0

56.5

5.8

5.0

16

37

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1117

2.4�

10�

457.0

56.5

5.8

5.0

12

25

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1123

9.4�

10�

12

57.0

56.5

5.8

5.0

18

38

A,B

,G,I

C,D

,E,F

,H,J

,K,L

1110

3.7�

10�

10

57.0

56.9

5.8

5.3

14

27

C,D

,E,F

,G,H

,I,J

,K,L

A,B

1118

7.1�

10�

21

57.0

56.7

5.8

5.5

27

50

B,C

,D,E

,F,G

,H,J

,K,L

A,I

1126

6.0�

10�

29

57.0

57.0

5.8

5.8

28

55

C,D

,H,J

,K,L

A,B

,E,F

,G,I

1141

2.8�

10�

16

57.0

56.3

5.8

6.3

23

49

D,H

A,B

,C,E

,F,G

,I,J

,K,L

Mu

rein

tran

sgly

cosy

lase

Q71W

Q6

EC

,C

W2078

5.8�

10�

329.0

27.8

5.5

4.8

947

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2073

4.9�

10�

729.0

27.6

5.5

4.9

14

61

A,B

,E,F

,G,I

C,D

,H,J

,K,L

2133

2.7�

10�

329.0

26.2

5.5

5.0

12

47

C,D

,H,J

,K,L

A,B

,E,F

,G,I

2086

4.4�

10�

229.0

26.5

5.5

5.0

938

C,D

,H,J

,K,L

A,B

,E,F

,G,I

2142

2.1�

10�

229.0

26.0

5.5

4.9

843

C,D

,H,J

,K,L

A,B

,E,F

,G,I

Pen

icil

lin

-bin

din

gp

rote

in,

tran

spep

tid

ase

/cell

div

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np

rote

inFts

I

Q8Y

763

EC

762

8.9�

10�

10

79.9

68.8

7.5

5.0

23

34

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

763

7.1�

10�

979.9

68.8

7.5

5.1

23

36

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Pen

icil

lin

-bin

din

gp

rote

in,

tran

spep

tid

ase

/cell

div

isio

np

rote

inFts

I

Q71X

X4

721

4.5�

10�

281.9

69.6

6.6

5.4

13

25

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

772

6.0�

10�

281.9

68.8

6.6

5.2

610

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Proteomics 2009, 9, 3136–3155 3145

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le2.

Co

nti

nu

ed

Pro

tein

desc

rip

tio

nU

niP

rotK

BID

a)

Pre

dic

ted

sub

cell

ula

rlo

cali

zati

on

b)

Sp

ot

E-v

alu

ec)

MM

(kD

a)d

)p

IP

ep

tid

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atc

hin

gS

eq

uen

ceco

vera

ge

e)

L.

mo

no

cyto

gen

es

stra

inf)

Th

eo

Exp

Th

eo

Exp

Pre

sen

ceA

bse

nce

Pen

icil

lin

-bin

din

gp

rote

in,

tran

spep

tid

ase

Q8Y

9I8

EC

1695

5.0�

10�

244.4

35.6

9.2

6.8

10

30

C,D

,H,J

,K,L

A,B

,E,F

,G,I

1669

4.4�

10�

244.4

35.6

9.2

7.2

820

A,C

,E,F

,H,K

,LB

,D,G

,I,J

1665

5.9�

10�

444.4

35.6

9.2

5.5

721

A,B

,G,I

C,D

,E,F

,H,,

K,L

1677

5.8�

10�

344.4

35.6

9.2

7.8

12

24

A,F

,HB

,C,D

,E,G

,I,J

,K,L

1676

6.3�

10�

444.4

35.6

9.2

6.9

11

34

IA

,B,C

,D,E

,F,G

,H,J

,K,L

Bif

un

ctio

nal

tran

sgly

cosy

lase

/tr

an

spep

tid

ase

pen

icil

lin

-b

ind

ing

pro

tein

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bp

A

Q71Y

C3

510

1.0�

10�

390.8

77.5

8.6

6.0

15

16

A,B

,E,F

,G,I

C,D

,H,J

,K,L

Cell

-en

velo

pe-r

ela

ted

tran

scri

pti

on

al

att

en

uato

rQ

8Y

9T

0E

C2257

2.0�

10�

534.1

22.6

9.1

5.1

13

32

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2261

1.9�

10�

834.1

22.6

9.1

5.5

13

39

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Cell

-en

velo

pe-r

ela

ted

tran

scri

pti

on

al

att

en

uato

r,LytR

Q8Y

4D

2E

C1942

7.4�

10�

439.1

33.0

5.6

4.6

925

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1989

1.9�

10�

239.1

32.2

5.6

4.6

10

28

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1942

7.4�

10�

439.1

33.0

5.6

4.6

925

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Deg

rad

ati

ve

en

zym

es

Pep

tid

ase

M23B

fam

ily

Q8Y

AE

4E

C,

MB

1538

2.5�

10�

444.4

44.4

9.0

9.0

10

28

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

3748

4.7�

10�

244.4

22.6

9.0

6.2

921

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Pep

tid

ase

M23B

fam

ily

Q71W

S4

EC

1333

3.2�

10�

4�

47.0

51.0

6.3

5.5

815

C,D

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,K,L

A,B

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,G,I

1334

6.3�

10�

247.0

51.1

6.3

5.7

825

A,B

,E,F

,G,I

C,D

,H,J

,K,L

1326

6.3�

10�

347.0

51.0

6.3

6.0

933

A,E

,F,I

B,C

,D,G

,H,J

,K,L

Ch

itin

ase

,C

hiA

Q71Y

D3

EC

1679

6.3�

10�

337.9

35.4

6.3

4.9

723

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1739

7.0�

10�

637.9

35.0

6.3

5.2

11

43

LA

,B,C

,D,E

,F,G

,H,I

,J,K

Cell

-su

rface

com

ple

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5C

W2168

1.7�

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2�

24.3

24.1

4.6

4.4

419

A,B

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,F,G

,H,I

,K,L

C,J

2094

9.4�

10�

324.3

27.6

4.6

4.3

637

C,J

A,B

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,H,I,K

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ran

sp

ort

an

dm

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bra

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bio

en

erg

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stem

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mp

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t,O

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Q9LA

T7

MB

890

1.7�

10�

262.5

63.5

5.3

4.8

11

28

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

AB

C-t

yp

ed

ipep

tid

etr

an

spo

rtsy

stem

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lute

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din

gp

rote

inco

mp

on

en

t

Q8Y

AJ0

MB

1145

7.8�

10�

758.0

55.7

5.0

4.7

16

40

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

AA

3-6

00

qu

ino

lo

xid

ase

sub

un

itII

,Q

oxA

Q8Y

AV

0M

B1922

5.1�

10�

241.5

33.2

6.0

5.0

11

30

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1947

1.3�

10�

341.5

32.8

6.0

5.6

833

B,C

,G,H

,J,K

,LA

,D,E

,F,I

1931

1.9�

10�

241.5

33.2

6.0

5.1

514

C,E

,F,H

,I,K

A,B

,D,G

,J,L

1938

5.9�

10�

241.5

41.8

6.0

5.2

723

A,B

,D,G

,IC

,E,F

,H,J

,K,L

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facto

ran

dvit

am

inb

iosyn

thesis

Am

ino

deo

xych

ori

sm

ate

lyase

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qzC

Q8Y

7E

9E

C3001

2.6�

10�

517.8

12.0

9.1

9.6

11

59

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

2917

7.9�

10�

417.8

13.6

9.1

7.8

947

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

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Q8Y

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EC

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P1581

2.4�

10�

936.3

36.5

5.2

4.9

13

42

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1585

1.3�

10�

336.3

36.9

5.2

5.0

12

30

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1582

4.8�

10�

236.3

36.2

5.2

5.0

12

32

A,B

,D,E

,F,G

,H,I

,J,K

,LC

En

ola

se,

En

oQ

71W

X1

EC

,C

P3745

6.0�

10�

17

46.5

47.1

4.7

4.5

21

54

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

3146 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le2.

Co

nti

nu

ed

Pro

tein

desc

rip

tio

nU

niP

rotK

BID

a)

Pre

dic

ted

sub

cell

ula

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cali

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on

b)

Sp

ot

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(kD

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862

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P1109

9.8�

10�

249.5

56.5

5.2

5.0

717

A,B

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,E,F

,G,H

,I,J

,K,L

1108

3.7�

10�

249.5

56.5

5.2

5.0

717

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Ph

osp

ho

gly

cera

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gk

Q4E

EP

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CP

1482

1.4�

10�

242.0

45.8

5.0

4.8

936

A,B

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Q71W

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P1021

4.7�

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956.1

59.3

5.2

4.9

17

40

A,B

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C,D

,H,J

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1020

6.8�

10�

356.1

15.3

5.2

4.5

719

A,C

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no

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CP

3749

2.7�

10�

219.3

15.3

4.7

4.5

756

A,B

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,G,H

,I,J

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bra

ne

sulf

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se,

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ted

Q8Y

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MB

1214

1.0�

10�

274.7

52.6

6.0

4.9

11

14

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1242

9.5�

10�

874.7

53.0

6.0

4.8

14

19

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1233

7.5�

10�

13

74.7

53.0

6.0

4.8

14

19

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

1248

9.5�

10�

10

74.7

53.0

6.0

4.8

18

32

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Ch

ap

ero

nin

Gro

ES

Q9A

GE

7E

C,

CP

3173

6.0�

10�

610.0

10.0

4.6

4.6

882

A,B

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,E,F

,G,H

,I,J

,K,L

Ch

ap

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nin

Gro

EL

Q71X

U6

EC

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P937

1.3�

10�

257.3

60.0

4.7

4.6

14

28

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

733

2.6�

10�

11

57.3

69.0

4.7

4.6

21

39

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Cla

ssI

heat-

sho

ckch

ap

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ne

pro

tein

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naK

Q9S

5A

4E

C,

CP

826

1.4�

10�

266.1

66.0

4.6

4.6

924

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

3750

6.6�

10�

11

66.1

68.0

4.6

4.5

22

44

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

666

9.1�

10�

10�

66.1

71.0

4.6

4.5

917

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Gen

era

lst

ress

pro

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Ctc

Q8Y

AD

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C,

CP

2216

1.5�

10�

322.6

22.6

4.4

4.5

643

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Co

ldsh

ock

pro

tein

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spA

Q71Z

V8

EC

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P3248

5.8�

10�

27.3

9.5

4.5

4.5

474

A,B

,C,D

,E,F

,G,H

,I,J

,K,L

Sta

ge

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pro

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Q724L7

EC

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P3033

6.4�

10�

311.2

11.2

4.5

4.5

956

A,B

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,E,F

,G,H

,I,J

,K,L

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sp

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Pro

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of

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fun

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3W

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W1951

6.2�

10�

234.6

33.0

5.0

4.6

626

A,B

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,E,F

,G,H

,I,J

,K,L

Sec-

tran

slo

cate

dp

rote

ino

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ow

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ion

Q8Y

5B

1E

C3201

2.7�

10�

213.0

9.9

9.6

10.0

638

A,B

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,E,F

,G,H

,I,J

,K,L

Ser/

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rp

rote

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LP

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724S

5C

W629

1.9�

10�

982.2

73.0

4.9

4.6

22

41

A,B

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Proteomics 2009, 9, 3136–3155 3147

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

predicted as cell-wall attached in the extracellular milieu,

namely, proteins with LPXTG, GW, LysM or WXL motifs.

Among the six L. monocytogenes murein-hydrolase autolysins

characterized to date, namely, Iap, P45 (protein of 45 kDa),

amidase, muramidase A (MurA), autolysin and Lmo0327

[74, 87], P45 (Table 2 and Supporting Information Fig. 1S;

Spots 1484, 1485, 1489, and 1505) and MurA (Table 2 and

Supporting Information Fig. 1S; Spot 1601) along with Iap

could be identified here as core constituents of L. mono-cytogenes exoproteome. As Iap, MurA, which is also called

NamA (N-acetylmuramidase A), is secreted in a SecA2-

dependent manner [88, 89]. Similarly, cell-wall hydrolase

Q8Y707 (Table 2 and Supporting Information Fig. 1S; Spots

1428, 1435, 1436, 1438, 1444, 1455 and 2103) was newly

identified as expressed and as part of the core exoproteome

of L. monocytogenes, together with a muramidase Q71W17

(Table 2 and Supporting Information Fig. 1S; Spots 1110,

1117, 1118, 1122, 1123, 1126 and 1141) specifically involved

in the flagellum assembly (COG1705: E-values 5 1.00

� 10�51) and a murein transglycosylase Q71WQ6 (Table 2

and Supporting Information Fig. 1S; Spots 2073, 2078,

2086, 2133 and 2142) involved in the recycling of muro-

peptides during cell elongation and/or division (IPR010611,

PF06725: E-values 5 1.5� 10�35; COG2821: E-value 5 7.00

� 10�05). Conversely, autolysin Q4EHT4 (Table 3,

Supporting Information Fig. 1S; Spot 3253) and flagellum-

specific muramidase Q8Y572 (Table 3 and Supporting

Information Fig. 1S; Spots 1726 and 1727) were only

expressed in a subset of L. monocytogenes strains. Besides,

these murein hydrolases involved in several degradation

processes related to cell-wall turnover, peptidoglycan

maturation and cell division, four penicillin-binding

proteins (Q8Y763, Q71XX4, Q8Y9I8 and Q71YC3) as well as

two cell-envelope-related transcriptional attenuators

(Q8Y9T0 and Q8Y4D2) were part of the core exoproteome

(Table 2) and were related to peptidoglycan biosynthesis for

cell-wall formation in the course of cell division. Two of

them (Q8Y763 and Q71YC3) were neither predicted nor

identified as part of the exoproteome in previous secretomic

investigation of L. monocytogenes [19]. Altogether, this indi-

cates that the cell wall is the siege of high activity, which can

have consequences on numerous cellular processes includ-

ing cell growth, biofilm formation, genetic competence,

protein secretion and pathogenicity [74].

3.2.3 Other degradative enzymes

Besides cell-wall hydrolases, several secreted degradative

enzymes could be identified here in L. monocytogenes culture

supernatants. Two peptidases of the M23B family (Q8YAE4

and Q71WS4), i.e. metallopeptidases belonging to the

MEROPS peptidase family clan M subtype B [90], were

identified in all the strains investigated (Table 2). On the

contrary, a D-alanyl-D-alanine carboxypeptidase A Q9ZIC4

(Table 3 and Supporting Information Fig. 1S; Spot 1437), i.e.

a serine peptidase belonging to the MEROPS peptidase

family S11 clan SE, and a serine protease Q71YE5 (Table 3

and Supporting Information Fig. 1S; Spot 1339), most

probably belonging to the MEROPS peptidase family S1 and

the peptidase family S6 (SSF50494: E-values 5 1.7� 10�22;

PF00089: E-values 5 8.2� 10�05), were only identified in a

limited number of the 12 L. monocytogenes strains investi-

gated. Peptidases can be involved in a wide variety of

biological processes from protein degradation to cell-wall

biogenesis as well as bacterial virulence [91]. Besides these

peptidases, a chitinase Q71YD3 encoded by chiA gene was

also identified extracellularly (Table 2) [92]. In the natural

environment, chitin is the second most common carbohy-

drate after cellulose and a major source of carbon and

nitrogen [93, 94]. As a component of the core exoproteome,

the chitinolytic activity of L. monocytogenes is most certainly

involved in supporting a saprophytic lifestyle in soil and

sediments. However, a dual role cannot be excluded as

observed in Legionella pneumophila, where ChiA is involved

in bacterial pathogenesis as it promotes the persistence of

this pathogen in a mammalian host [95]. Similarly, CscB

Q8Y9E5 (Table 2 and Supporting Information Fig. 1S; Spots

2094 and 2168), which is described as putatively involved in

plant carbohydrate utilization as part of protein cell-surface

complexes [72], is also considered as a coaggregation-

promoting factor by mediating aggregation of other bacterial

species and might thus be of importance in the biofilm

formation process. Despite the prediction of CscB as an

extracellular protein, this protein was not identified in

previous investigation of L. monocytogenes exoproteome [19].

As already reported in Lactobacillus plantarum, CscB is non-

covalently bound to the bacterial cell surface as it can also be

found in the extracellular milieu.

3.2.4 Proteins secreted by unknown pathways

Several proteins lacking a putative N-terminal signal peptide

and with no legitimate protein secretion system explaining

for their final localization had, furthermore, no obvious

biological function in the extracellular milieu. These

proteins are primarily predicted as cytoplasmic, some of

them being first and foremost involved in the central

metabolism, such as glyceraldehyde-3-phosphate dehy-

drogenase or enolase (Table 2). However, the presence of

such proteins has been reported in the culture supernatant

of numerous bacterial species, including L. monocytogeneswhere they were in addition reported as also being localized

in the cell wall [19, 96]. Clearly, these proteins have multiple

final subcellular localizations. Among these proteins, cata-

lase and cysteine synthetase A appeared to be part of the

exoproteome only in a subset of L. monocytogenes strains

(Table 3). As further indicated by the presence of general

stress protein Ctc or cold shock protein CspA (Table 2), a

number of those proteins are primarily related to bacterial

stress resistance. Finally, this proteomic analysis also

3148 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le3.

Vari

an

texo

pro

teo

me

of

L.

mo

no

cyto

gen

es

speci

es

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ow

ing

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ule

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Ph

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2002

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1999

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C1935

4.4�

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430.6

33.0

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6.4

637

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2816

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415.5

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op

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fu

nkn

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ion

Q8Y

850

MB

2058

8.9�

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430.8

30.1

5.9

5.6

939

A,B

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2080

3.1�

10�

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28.2

5.9

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631

B,C

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,D,E

,F,I

Proteomics 2009, 9, 3136–3155 3149

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

revealed the expression of several proteins whose genes

were originally annotated as hypothetical. However, their

functions remain unclear as no further insight could be

gained from additional in silico analyses as described above.

Four of them (Q8Y3W5, Q8Y5B1, Q724S5 and Q8Y989)

were part of the core exoproteome (Table 2), whereas the

remaining six (Q8Y6D5, Q721A2, Q71XZ2, Q8Y850,

Q721L6 and Q8Y9F2) were part of the variant exoproteome

(Table 3). Altogether, six of these proteins exhibited an

N-terminal signal peptide and were thus more likely trans-

located in a Sec-dependent manner, including one lipopro-

tein Q8Y850 (Table 2) and one LPXTG protein Q8Y3W5

(Table 3). One of them (Q721A2) was neither predicted nor

identified as part of the exoproteome from previous secre-

tomic analysis of L. monocytogenes [19].

4 Concluding remarks

From the present investigation, a large majority of the

proteins identified within the extracellular milieu were

indeed predicted as extracellular, i.e. 51 out of 60 proteins

(85%). Most of them were actually predicted as secreted viathe Sec pathway, i.e. 42 out of 60 proteins (70%) (Fig. 3). By

comparison, exoproteomes from other Gram positive

bacteria such as Bacillus licheniformis [97] and Bacillus subtilis[98] revealed that 62% (89/143) and 53% (48/90) of the

identified proteins could be predicted as secreted. As for

L. monocytogenes, most of them contained a Sec-type signal

peptide (75/89 and 34/48, respectively) and were probably

exported by the Sec pathway. In B. licheniformis and

B. subtilis, respectively, 4 and 14 of the predicted secreted

proteins exhibited a potential twin-arginine signal peptide

suggesting they were directed to the Tat machinery. Among

the ten remaining proteins known to be secreted in

B. licheniformis, seven were flagellum proteins most prob-

ably transported via the FEA and three phage-related

proteins were presumably secreted via holins [97].

While the first exoproteomic analysis by Trost et al. [19]

had the merit of giving an initial view of what makes

L. monocytogenes singular from a non-pathogenic counter-

part, i.e. Listeria innocua CIP11262, as it highlighted that

such differences manifest the most in the exoproteome, this

view was also biased by the fact that it focused on only one

L. monocytogenes strain. The present investigation on the

exoproteomes of 12 different L. monocytogenes strains

allowed taking the biodiversity into account and getting a

more objective overview of the exoproteome from L. mono-cytogenes species. Among the 60 proteins identified here in

the exoproteome of the 12 L. monocytogenes strains investi-

gated, 41 were common to the 105 proteins identified by

Trost et al. [19]. Among them, the major virulence factors

ActA, Hly, PlcB, InlC were identified in all the strains here

investigated. On the other hand, PlcA, a protein involved in

the escape from the phagocytic vacuole, was absent from

one strain. Some proteins primarily predicted as cyto-Tab

le3.

Co

nti

nu

ed

Pro

tein

desc

rip

tio

nU

niP

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a)

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dic

ted

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ula

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cali

zati

on

b)

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ot

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(kD

a)d

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tid

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hin

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eq

uen

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mo

no

cyto

gen

es

stra

inf)

Th

eo

Exp

Th

eo

Exp

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sen

ceA

bse

nce

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rola

se,a/b

fold

fam

ily

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fun

ctio

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MB

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,E,F

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,I,K

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rmati

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ith

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res

est

ab

lish

ed

wit

ha

sig

nifi

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resh

old

set

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ag

ain

stth

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or

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icate

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eo

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eo

)an

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es.

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eo

reti

cal

avera

ge

mo

lecu

lar

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(MM

)ca

lcu

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du

sin

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ge

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valu

es

of

am

ino

aci

ds.

e)

Exp

ress

ed

as

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en

tag

e.

f)P

rese

nce

or

ab

sen

ceo

fth

ep

rote

insp

ot

inth

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mo

no

cyto

gen

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stra

ins

invest

igate

das

list

ed

inT

ab

le1.

3150 E. Dumas et al. Proteomics 2009, 9, 3136–3155

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

plasmic and with no protein secretion system explaining for

their final localization were found in the extracellular milieu

in our study as well as in the investigation of Trost et al. [19],

thus confirming their multiple subcellular localizations.

From this first investigation on the strain L. monocytogenesEGD-e, the maximum number of proteins present extra-

cellularly was predicted at 121. From our investigation, the

picture of L. monocytogenes proteins present extracellularly

has been broadened in relation to the protein secretion

systems involved [6]. Among the 60 exoproteins here iden-

tified 43 clearly appeared as part of the core exoproteome of

L. monocytogenes species and 17 were limited to a subset of

strains. Compared with Trost et al. [19], 19 proteins were

newly identified in our present investigation, including nine

proteins as part of the variant exoproteome.

While variations in the exoproteomes are linked to some

virulence factors, cell-wall hydrolases or degradative

enzymes, most of these proteins have unknown functions.

Considering the wide variety of biological processes in

which cell-wall hydrolases are involved, differences in their

expression from one L. monocytogenes strain to another

certainly result in the modification of their phenotypes for

example in terms of biofilm formation, genetic competence,

protein secretion or pathogenicity [74]. These potential

correlations between the presence of various cell-wall

hydrolases and a particular biological process should require

further investigation in order to gain insight into the

physiology of the L. monocytogenes species.

Among the 60 proteins identified extracellularly, 17 out

of 18 proteins primarily cytoplasmic were also predicted as

translocated by unknown secretion systems whereas the

remaining proteins were all predicted as secreted in a Sec-

dependent manner (Fig. 3 and Supporting Information

Table S1). As enolase, DnaK or EF-Tu, which were recently

demonstrated to bind human plasminogen [96], or glycer-

aldehyde-3-phosphate dehydrogenase, which was further

reported as interfering with Rab5a [99], some of these

listerial proteins with multiple subcellular localizations may

moonlight when present extracellularly and have a role in

bacterial virulence. In a previous investigation combining

two complementary techniques viz. 2-DE and HPLC-MS/

MS, 105 proteins could be identified as part of the exopro-

teome of L. monocytogenes EGD-e including 51 proteins

exhibiting no signal peptide and thus a specific mechanism

explaining their translocation was considered unknown [19].

However, it later appeared that some of these proteins were

most probably secreted via holins [6]. None of the proteins

identified in the present study and lacking a signal peptide

were predicted as substrates of the holins, FEA or Wss.

Some of these proteins might actually be secreted in a Sec-

dependent manner but in conjunction with SecA2, a para-

logue to the cytosolic ATPase SecA [6]. Indeed, SecA2 is

required for secretion of listerial FbpA and MnSod, both

lacking a putative N-terminal signal peptide [85, 100]. SecA2

was even confirmed as promoting the release of the heat

shock protein GroEL [89]. Alternatively, cell lysis could

explain the presence of these primarily cytoplasmic proteins

in the culture supernantant as suggested by the identifica-

tion of several cell-wall hydrolases. As reported in Strepto-coccus pneumoniae, however, rather than autolysis this could

result from allolysis, another kind of apoptotic mechanism

where competent cells triggered lysis of non-competent cells

in a tightly control process [101]. Such phenomenon could

explain the extracellular localization of primarily cyto-

plasmic proteins with no obvious mechanism of secretion,

though this possibility has not yet been questioned in

Figure 3. Schematic representation of protein secretion pathways predicted in Listeria spp [6]. Most of the proteins identified in the

exoproteome of the 12 L. monocytogenes strains investigated were predicted as secreted via the Sec pathway (70%), while all others were

predicted as translocated by unknown secretion systems (30%). Cyto: Cytoplasm; CM: Cytoplasmic Membrane; CW: Cell Wall; Ext:

Extracellular milieu; Sec: Secretion apparatus; Tat: Twin-arginine translocation; FPE: Fimbrilin-Protein Exporter; FEA: Flagella Export

Apparatus; Wss: WXG100 (proteins with WXG motif of �100 amino acyl residues) secretion system.

Proteomics 2009, 9, 3136–3155 3151

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

L. monocytogenes and undoubtedly requires further investi-

gations [6]. Eventually, it cannot be excluded that uncovered

secretion systems are present in L. monocytogenes.While this investigation gives the first picture of the core

and variant exoproteomes of L. monocytogenes species, this

comparative subproteomic analysis should be seen as an

initial statement giving a primal view of these exoproteomes

as the number of strains investigated was limited to 12.

Besides uncovering additional extracellular proteins, it can

be expected that the number of proteins presently described

as the core exoproteome decreases in favor of the variant

exoproteome as more extracellular proteomes from various

L. monocytogenes strains are deciphered. Ultimately, it could

establish to what extent the level of virulence of different

L. monocytogenes strains is correlated with the absence of

some extracellular virulence factors in the aim of uncovering

markers indicative of the biohazard of L. monocytogenesstrains isolated from food products [13, 102, 103].

This work received financial support from the ‘‘InstitutNational de la Recherche Agronomique’’ and the FrenchMinistere de l’agriculture et de la peche (DGAL N1 A03/02).Emilie Dumas is a PhD research fellow granted by the ‘‘Minis-tere de l’Education Nationale, de l’Education Superieure et de laRecherche’’. We are grateful to the INRA MIGALE bioinf-ormatics platform (http://migale.jouy.inra.fr) for providingcomputational resources.

The authors have declared no conflict of interest.

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