Comprehensive Appraisal of the Extracellular Proteins from a Monoderm Bacterium: Theoretical and Empirical Exoproteomes of Listeria monocytogenes EGD-e by Secretomics

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Comprehensive Appraisal of the Extracellular Proteins from a Monoderm Bacterium: Theoretical and Empirical Exoproteomes of Listeria monocytogenes EGD-e by Secretomics Mickae¨l Desvaux,*,† Emilie Dumas,†,‡ Ingrid Chafsey,† Christophe Chambon,§ and Michel He´braud†,§ INRA, UR454 Microbiology, Food Quality and Safety Team, F-63122 Saint-Gene`s Champanelle, France, and INRA, Metabolism Exploration Platform, Proteomics component, F-63122 Saint-Gene`s Champanelle, France Received April 23, 2010

Defined as proteins actively transported via secretion systems, secreted proteins can have radically different subcellular destinations in monoderm (Gram-positive) bacteria. From degradative enzymes in saprophytes to virulence factors in pathogens, secreted proteins are the main tools used by bacteria to interact with their surroundings. The etiological agent of listeriosis, Listeria monocytogenes, is a Gram-positive facultative intracellular foodborne pathogen, whose ecological niche is the soil and as such should be primarily considered as a ubiquitous saprophyte. Recent advances on protein secretion systems in this species prompted us to investigate the exoproteome. First, an original and rational bioinformatic strategy was developed to mimic the protein exportation steps leading to the extracellular localization of secreted proteins; 79 exoproteins were predicted as secreted via Sec, 1 exoprotein via Tat, 4 bacteriocins via ABC exporters, 3 exoproteins via holins, and 3 exoproteins via the WXG100 system. This bioinformatic analysis allowed for defining a databank of the mature protein set in L. monocytogenes, which was used for generating the theoretical exoproteome and for subsequent protein identification by proteomics. 2-DE proteomic analyses were performed over a wide pI range to experimentally cover the largest protein spectrum possible. A total of 120 spots could be resolved and identified, which corresponded to 50 distinct proteins. These exoproteins were essentially virulence factors, degradative enzymes, and proteins of unknown functions, which exportation would essentially rely on the Sec pathway or nonclassical secretion. This investigation resulted in the first comprehensive appraisal of the exoproteome of L. monocytogenes EGD-e based on theoretical and experimental secretomic analyses, which further provided indications on listerial physiology in relation with its habitat and lifestyle. The novel and rational strategy described here is generic and has been purposely designed for the prediction of proteins localized extracellularly in monoderm bacteria. Keywords: Gram-positive bacteria • Listeria monocytogenes • extracellular proteome • secreted protein • protein secretion system • secretome • MALDI-TOF mass spectrometry • bioinformatic analysis

Introduction As the main tools used by bacteria to interact with their environment, secreted proteins are relevant to the bacterial lifestyle.1,2 From degradative enzymes in saprophytes to virulence factors in pathogens, secreted proteins cover a vast variety of functions, sometimes essential for cell viability, and a crucial importance in ecological interactions.3 By definition, secreted * To whom correspondence should be addressed. INRA (French National Institute for Agronomical Research), Clermont-Ferrand Research Centre, UR454 Microbiology, Food Quality and Safety Team, Department of Microbiology and Food Chain, Site of Theix, F-63122 Saint-Gene`s Champanelle, France. Tel.: +33 (0)4 73 62 47 23. Fax: +33 (0)4 73 62 45 81. E-mail: [email protected]. † UR454 Microbiology, Food Quality and Safety Team. ‡ Present address: Universite´ Lyon 1, Laboratoire de Recherche en Ge´nie Industriel Alimentaire (LRGIA), EA 3733, IUTA, F-01000 Bourg en Bresse, France. § Metabolism Exploration Platform, Proteomics component.

5076 Journal of Proteome Research 2010, 9, 5076–5092 Published on Web 09/14/2010

proteins are proteins actively transported via secretion systems, but they can have radically different final destinations;4 in monoderm (Gram-positive) bacteria, secreted proteins can be (i) anchored to the cytoplasmic membrane, (ii) associated with the cell wall, (iii) released into the extracellular milieu, or (iv) injected into a host cell. Final subcellular locations of these secreted proteins actually rely on the presence of various secondary structures and/or domains (motifs) that can retain the post-translationally modified proteins within the cell envelope of monoderm bacteria.5 In contrast, the subset of proteins localized in the extracellular milieu constitutes the extracellular proteome, also called exoproteome.6 In any case, the gating systems involved in the secretion of such effectors remain a key event in the maturation process, which as a whole corresponds to the secretome.4 In monoderm bacteria, seven protein secretion systems are currently recognized, namely (i) the Sec (Secretion) pathway, (ii) the Tat (Twin-arginine trans10.1021/pr1003642

 2010 American Chemical Society

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Comprehensive Exoproteome of L. monocytogenes EGD-e location) pathway, (iii) the FPE (Fimbrilin-Protein Exporter), (iv) some ABC (ATP-binding cassette) protein exporters, (v) the FEA (Flagellum Export Apparatus), (vi) the holins (holeforming), and (vii) the Wss (WXG100 secretion system).4,7,8 Not all of the secretion pathways are systematically present in a single organism, and their respective contribution in transport of exoproteins greatly varies from one organism to another.9 Listeria monocytogenes is an ubiquitous aeroanaerobic bacterium belonging to the phylum Firmicutes, class Bacilli, order Bacillales, family Listeriaceae.10 Considering the Gram-staining result, cell-envelope architecture, and taxonomic group, L. monocytogenes is a Gram-positive bacterial species in all three meanings of the term.4 L. monocytogenes is generally described as the etiological agent of listeriosis, a quite rare but serious foodborne infection essentially for immunocompromised people, where it can be lethal in a significantly high number of cases. The infectious cycle of this opportunistic pathogen is now well established, ranking L. monocytogenes among the best model of host-pathogen interactions and intracellular parasitism.11 After ingestion of contaminated foodstuff, L. monocytogenes multiplies in the digestive tract, crosses the intestinal barrier, and disseminates to deeper organs resulting ultimately in brain and materno-feotal infections. The pathogenicity of L. monocytogenes is associated with the production of defined virulence factors.12 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 via phagocytosis. Then, released listeriolysin O (LLO) and phospholipases C (PlcA and PlcB) enable escape from the phagocytic vacuole, whereas membrane-anchored ActA (actin assembly) is responsible for actin-based motility allowing for cell-to-cell spread. Thus, as in any virulent bacteria, listerial pathogenicity depends greatly on the ability to secrete virulence factors, which are displayed on the bacterial cell surface, released into the extracellular milieu, or even injected directly into the host cell.13 Until recently, an overview of the protein secretion systems present in L. monocytogenes remained elusive. Following genomic analysis, all seven protein secretion systems recognized in monoderm bacteria were uncovered in L. monocytogenes.14,15 Using seven different bioinformatic tools, a total number of 121 proteins were predicted as released into the extracellular milieu.16 Compared to early genomic analysis where the number of exoproteins was estimated at 86,17 this latter investigation allowed for improving of the prediction of exoproteins in this strain both quantitatively and qualitatively.16 Unfortunately, at the time of this investigation, protein secretion systems present in L. monocytogenes were not known and could not then be taken into account. As a result, all exoproteins were predicted as secreted via Sec pathway. Moreover, in the meantime, several new bioinformatic tools became available for prediction of the final location of bacterial proteins.18 Considering L. monocytogenes pathogenicity manisfests the most in the exoproteome,16 we recently investigated this subproteome in 12 representative strains of the species from different origins, serovars, and virulence levels.19 This approach allowed for providing a first definition of the core and variant exoproteomes of the species and the identification of potential markers for serotyping, origin of the isolates, and risk analysis.20,21 While this study was considering the biodiversity of the species, the limit resided in the genome sequences availability of the investigated L. monocytogenes strains. Altogether, this prompted us to reinvestigate and focus on the exoproteome of L. monocytogenes sequenced strain EGD-e.

Considering that genomic identification of exoproteins was previously and simply based on the presence of a signal peptide, where the output was just cleared off from proteins reported with cell-envelope retention domains,16 we developed an original and rational bioinformatic strategy that mimisc the protein secretion steps leading to the extracellular localization (GO: 0005576) of secreted proteins. Compared with our previous work where an in silico approach was designed to analyze proteomic data and support identification of proteins present in culture supernatant,19 the present strategy was improved with additional and most up-to-date prediction tools as well as the development of a rational workflow that could be fuelled with genomic data, whose results were integrated in a consensus view for predicting exoproteins and were processed to define a databank of the mature protein set in L. monocytogenes EGD-e. This databank was further used for generating a theoretical map of the exoproteome and for subsequent proteomic analysis, especially protein identification by peptide mass fingerprinting. Furthermore, to cover a large protein set, 2-DE proteomic analyses were directly performed in wide pI range, that is, 3-10. The present investigation results in the first comprehensive appraisal of the exoproteome of L. monocytogenes EGD-e taking into account both the secretion systems and the secreted proteins, which truly corresponds to a secretomic analysis. The general strategy we described has been purposely designed for the prediction of proteins localized extracellularly in other monoderm bacteria.

Experimental Procedures Bioinformatic Analyses for Predicting Extracellular Proteins. Bioinformatic analyses were performed by web-based servers or under Linux environment and Sun Grid Engine (SGE) from Topaze server homed at INRA MIGALE bioinformatics platform from MIG (Mathe´matique, Informatique et Ge´nome) Research Unit (INRA, Jouy-en-Josas, France). The complete genome, coding sequences (CDS), and annotation files for L. monocytogenes EGD-e were downloaded from GenBank (ftp: //ftp.ncbi. nih.gov/genbank/genomes/Bacteria/Listeria_monocytogenes/). Genomic analysis was performed using previously described bioinformatic tools.19 Positive predictions for N-terminal signal peptides, transmembrane R-helices, and protein subcellular locations were combined into a majority vote decision. N-terminal signal peptides (11-60 amino acid residues) were predicted combining (i) SignalP v2.0 and v3.0 using NN and HMM,22 (ii) SPScan,23,24 (iii) Phobius,25 (iv) SOSUIsignal,26 (v) PSORTb v2.0.4,27,28 PrediSi,29 and (vi) Signal-3 L.30 Tat signal peptide prediction was performed from TatP v1.031 and TATFIND v1.432 where both RR and KR motifs were taken into consideration. Whenever possible, all of these tools were trained on prokaryotes, bacteria, or Gram-positive bacteria with truncation set either at 35, 70, 140 or disabled. Pseudopilinlike signal peptides were searched for consensus motif [AG]F[TS]LX[EF] as previously described.19,15 Signal peptides of protein substrates of ABC exporters, namely bacteriocins, were identified using BAGEL.33 Lipobox were found using DOLOP,34 LipoP v1.0,35 SPEPLip,36 LipPred37 and scanned by ScanProsite38 with both PS51257 profile and G+LPP v2.0 pattern, i.e. [MV]-X(0,13)-[RK]-{DERKQ}(6,20)-[LIVMFESTAGPC]-[LVIAMFTG]-[IVMSTAGCP]-[AGS]-C,39 as well as output from the Lipo-HMM specifically developed by Baumga¨rtner et al.40 Nonclassically secreted proteins were identified from SecretomeP v2.0 trained on Gram-positive bacteria41 but also some support vector machines (SVMs) described below. Journal of Proteome Research • Vol. 9, No. 10, 2010 5077

research articles Transmembrane R-helices were predicted combining (i) TMHMM v2.0,42 (ii) SVMtm v1.043 (iii) THUMBUP v1.0,44 (iv) SOSUI v1.10,45 (v) HMMTOP v2.0,46 (vi) PHDhtm v1.0,47 (vii) UMDHMMTMHP v1.0,44 and (viii) MEMSAT v3.0.48 The presence of TMDs in signal peptides (i.e., the H-domain) and LPXTG motifs were carefully taken into consideration for the prediction of integral membrane proteins (IMP). Cell wall attachment domains were searched from InterProScan v4.2, HMMER v2.3.2 for HMM,49 RPS-BLAST v2.2.19 (Reverse Position-Specific BLAST)50 or ScanProsite. Databases used were InterPro (IPR) v4.3,51 Pfam (PF) v21.0,52 SMART (SM) v5.1,53 TIGRfam (TIGR) v6.0,54 SuperFamily (SSF) SCOP v1.71,55,56 PRK v3.0,57 PIRSF,58 COG v1.059 and Prosite (PS) v20.7.60 Search for LPXTG motif also involved the use of LPXTG-HMM profile specifically developed by Boekhorst et al.61 WXL domain was searched using the [LI][TE]W[TS]L motif as previously described.19,62 Position-specific iterated BLAST (PSI-BLAST v2.2.20)50 searches were executed when needed against UniProtKB v15.3 until convergence.63 All searches were performed with E-value cutoff set at 10-3. SVMs were used to complete subcellular location of proteins, that is, SubLoc v1.064 and LocTree65 trained on prokaryotes where only extracellular and cytoplasmic subcellular locations are considered, whereas CELLO v2.566 and PSORTb v2.0.4 trained on Gram-positive bacteria28 considered extracellular, cell wall, membrane, and cytoplasmic subcellular compartments as well as multiple location sites. Proteomic Analysis of Extracellular Proteins. L. monocytogenes EGD-e was grown until early stationary phase as previously described.19-21,67 Supernatant was recovered from bacterial culture following centrifugation and filter sterilization in the presence of proteases inhibitor. To evaluate potential cell lysis, specific enzyme activity of aminopeptide C was assayed as previously described.68 After protein precipitation in the presence of sodium deoxycholate and cold TCA, the pellet obtained after centrifugation was washed with ice-cold acetone and solubilized in IEF buffer. 2-DE was performed as previously described.19 Briefly, 17 cm nonlinear pH 3-10 IPG strips were passively rehydrated prior to isoelectric focusing. The second dimension was carried out with 12.5% acrylamide before the gels be stained, scanned, and analyzed using Image Master 2D Platinum software v5.0 (GE Healthcare). Extracellular proteins were extracted from two independent cultures and five 2-DE gels per protein samples were performed. For protein identification by MALDI-TOF mass spectrometry, protein spots were first excised, destained, and submitted to tryptic digestion.69 Positive ion MALDI mass spectra were recorded in the reflectron mode of a MALDI-TOF MS (Voyager DE-Pro, Perseptive BioSystems) using DataExplorer v4.0 software for data collection and analysis. Monoisotopic peptide masses were assigned and used for database searches with the Mascot v2.2.0 software. Interrogations were performed against a database containing 2846 distinct protein entries corresponding to the mature set of L. monocytogenes EGD-e, that is, DBMature-LmoEGDe. The following parameters were considered for the searches: a maximum ion mass tolerance of (25 or 50 ppm, possible modification of cysteines by carbamidomethylation, as well as partial oxidation of methionine.

Results Secretomic Analysis of Extracellular Proteins in L. monocytogenes EGD-e. In Listeria, 7 secretion systems allow translocation of genome encoded polypeptides through the cytoplas5078

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mic membrane. However, FEA and FPE are respectively involved in flagellum and competence pseudopilus assembly.70,71 Moreover, Sec-secreted proteins can be14 (i) anchored to the cytoplasmic membrane, (ii) associated with the cell wall, or (iii) released into the extracellular milieu. On the basis of current knowledge in the field, we applied a rational strategy mimicking as close as possible the molecular steps encountered by a protein in the course of secretion to estimate the number of extracellular proteins in L. monocytogenes EGD-e (Figure 1). This approach was formalized with a bioinformatic workflow based on the application of several predictive tools (Figure 2). Detailed results of the bioinformatic analyses are provided as Supporting Information in Table 1S. Prediction of Exoproteins Encoded on the L. monocytogenes EGD-e Genome. The 2846 CDS present in L. monocytogenes EGD-e chromosome were first screened for the presence of N-terminal signal peptide (Supporting Information Table 1S). Besides Sec translocon, N-terminal signal peptide targeting proteins to Tat, FPE and ABC pathways were also considered. Two proteins exhibiting a Tat signal peptide and 5 proteins with signal peptides specific to bacteriocin/pheromone secreted via ABC exporters were uncovered together with 5 prepseudopilins bearing Type 4 prepilin-like signal peptides, no longer considered as part of the exoproteome. Proteins lacking an N-terminal signal peptide were then scanned for alternative protein secretion pathways. Three proteins were identified as secreted via holins and 3 others via Wss. Proteins targeted to the FEA were removed from the output. All proteins were further analyzed for the presence of transmembrane R-helices (Supporting Information Table 1S). Proteins with a predicted N-terminal signal peptide were further screened using SecretomeP to eliminate false-positive, in particular single-spanning membrane proteins of Type II, where uncleaved signal peptide serves as signal-anchor sequence.6,18 All identified IMPs were removed from the output. Proteins were further scanned for the presence of cell-envelope binding domains and removed from the output (Supporting Information Table 1S). For GW proteins though, 4 of them were considered as extracellular rather than attached to the bacterial cell surface as they exhibited only one GW module.72,73 To backup prediction of extracellular location but also considering that automatic annotations may give false functional prediction74 and that a high proportion of genes (about 79% of the CDS) were originally annotated as hypothetical in L. monocytogenes EGD-e genome in GenBank,17 all the annotations were verified seeking for matches against various databases. Proteins resulting in inconclusive or no matches were further PSIBLASTed against UniProtKB database until convergence. Besides refining prediction results and improving the original annotation, the number of exoproteins with unknown function was then reduced from 76 to 29 (Supporting Information Table 1S). In the end, 90 exoproteins were predicted as transported via protein secretion systems and localized extracellularly (GO: 0005576), including 79 proteins via Sec, 1 protein via Tat, 4 bacteriocins via ABC protein exporters, 3 proteins via holins, 3 proteins via Wss. Predicted preproteins secreted via Sec exhibited N-terminal signal peptides from 18 to 58 amino acids, with a modal value (mode) of 29 residues (Figure 3). Interestingly, 88% of the predicted signal peptides possess at least one helix-breaking residue, that is, proline or glycine, within the h-domain (Supporting Information Table 1S). When present, especially toward the end of the h-domain, these residues

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Figure 1. Schematic representation of the rationale for exoprotein prediction in Gram-positive bacteria. This strategy can be summarized as the answering to 5 successive questions: (1) the presence of an N-terminal signal peptide, (2) the type of signal peptide, (3) the secretion system used, (4) the presence of transmembrane domain(s), and (5) the presence of cell-envelope binding domain(s). Signal peptides were subdivided into Sec-dependent, Tat-dependent, FPE-dependent, and ABC-dependent ones. While the type of signal peptide allowed us to attribute a specific secretion system, proteins lacking a signal peptides were divided into those secreted in a FEA-, holin-, Wss-dependent manner or alternatively via nonclassical secretion; it can be stressed that some proteins lacking a signal peptide can nonetheless be translocated via Sec, that is, in a SecA2-dependent manner. As proteins secreted via FEA are components of the flagella (A) and those secreted via FPE are involved in the formation of competence pseudopilus (B), they were not predicted as present in the extracellular milieu. Sec-dependent signal peptides were screened for the presence of a lipobox, indicating they were lipoproteins anchored to plasma membrane (C) and proteins exhibiting noncovalent cell wall binding motifs in L. monocytogenes, that is, WXL (D), LysM (E), or more than one GW (F) or a covalent cell wall binding motif, that is, LPXTG (G) were removed from the output. From the 2846 CDS in L. monocytogenes EGD-e, 90 proteins were thus predicted as secreted via Sec, Tat, ABC, holins, or Wss pathways in the extracellular milieu (GO: 005576). Additionally, 215 proteins primarily considered as cytoplasmic were also predicted as potentially extracellularly localized and secreted via nonclassical pathway(s) (not reported on figure). CDS, coding sequences; SP, signal peptide; Sec, secretion; Tat, twin-arginine translocation; FPE, fimbrilin-protein exporter; ABC, ATP-binding cassette exporter; FEA, flagella export apparatus; holin, hole forming; Wss, WXG100 secretion system; Cyto, cytoplasm; CM, cytoplasmic membrane; CW, cell wall; EM, extracellular milieu; GO, gene ontology.

facilitate the cleavage by signal peptidase I (SPase I).75 Besides proteins translocated via Sec, Tat, ABC, holins or Wss pathways, 215 proteins were predicted as extracellularly localized following nonclassical secretion.41,76 Some of these proteins lacking a signal peptide could be secreted in a route involving the alternative cytosolic ATPase SecA2, a paralogue of SecA, that most certainly converge to the Sec translocon in Gram-positive bacteria.15,77 Nonetheless, the prediction of nonclassically secreted proteins must be considered with caution, especially in a context of pure genomic analysis, as false positives cannot be excluded.6 From this genomic analysis, the mature set of exoproteins could be further deduced by removing predicted N-terminal signal peptides from the preprotein sequences leading to the databank DB-Mature-LmoEGDe, which served for generating a theoretical 2-DE map of the extracellular proteome (Figure 4 and Table 1). Exoproteins Secreted via Sec Pathway. The 79 CDS encoding proteins predicted as secreted in a Sec-dependent manner represented the largest set of extracellular proteins. Despite interrogations of conserved-motif databases as well as PSIBLAST searches, no function could be predicted for 30 of them. Key virulence factors listeriolysin O (LLO), phosphatidylinositol

phospholipase C (PlcA) and phosphatidylcholine phospholipase C (PlcB) were indeed predicted as secreted extracellularly14 (Tables 1 and 1S, Supporting Information). In addition, internalin C (InlC) belongs to the third and last subfamily class of internalin,78 but its role in virulence remains unclear.79 Conversely, the activity of MnSOD post-translationally regulated by phosphorylation is critical in L. monocytogenes pathogenesis.80,81 Several degradative enzymes were related to carbohydrate catabolism (Tables 1 and 1S, Supporting Information). Lmo1913 (IPR008928: E-value)1.5 × 10-18) belonged to the six-hairpin glycosidase family, which includes various glucoamylases and cellulases. Lmo2444 (COG1501: E-value ) 1.0 × 10-161) bore two galactose-binding like domains (IPR008979: 5.1 × 10-23 e E-values e 5.7 × 10-14) and belonged to the glycosyl hydrolase family 31. Chitinases ChiA and ChiB as well as Lmo2467 exhibiting a chitin-binding domain were dedicated to chitin catabolism.82 In the natural environment, chitin is the second most common carbohydrate polymer after cellulose and a major source of carbon and nitrogen.83,84 Chitinolytic activity of L. monocytogenes is most certainly involved in supporting a saprophytic lifestyle in soil and sediments. This phenotype may Journal of Proteome Research • Vol. 9, No. 10, 2010 5079

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Figure 2. Bioinformatic workflow based on the rational and original strategy mimicking the protein secretion steps followed by extracellular proteins in Gram-positive bacteria. The flowchart derives from the rationale depicted in Figure 1 and indicates the sequential use of the different bioinformatic tools. Detailed description of the bioinformatic approach is provided in the text. Briefly, the CDS were screened for the presence of signal peptide, where signal peptide specific to Tat, FPE, and ABC exporters were further scanned. CDS exhibiting no signal peptide were screened as potential substrates for alternative secretion systems using available bioinformatic tools and internal databases based on literature survey.15 Proteins predicted as secreted were then scanned for the presence of hydrophobic R-helical transmembrane domain(s), cytoplasmic-membrane anchoring motif and cell-wall anchoring domain(s). Proteins exhibiting cell-envelope retention domain(s) were cleared from the output, which resulted in the predicted exoproteins. Functional annotations of these exoproteins were finally checked to backup location prediction. This genomic analysis further allowed to attribute a secretion system to each of the exoproteins. This bioinformatic strategy is generic and can be applied as such using CDS from any Gram-positive bacteria. CDS, coding sequences; SP, signal peptide; DB, database; Sec, secretion; Tat, twin-arginine translocation; FPE, fimbrilin-protein exporter; ABC, ATP-binding cassette exporter; FEA, flagella export apparatus; Wss, WXG100 secretion system; NC, nonclassical secretion; Y, yes; N, no.

also have an impact in food safety considering chitin enhances the survival as well as the biocide resistance.85,86 In addition, several exoproteases and exopeptidases encoded by genes originally annotated as hypothetical were revealed (Tables 1 and 1S). This included serine protease Lmo2056 (IPR014044: E-value ) 2.4 × 10-22) as well as metalloproteases Lmo1264 and Lmo1654 (SSF55486: 7.3 × 10-17 e E-values e 8.1 × 10-16). Most uncovered peptidases were serine peptidases either belonging to families S9 (Lmo2074; PSI-BLAST: 2.0 × 10-68 e E-values e 2.0 × 10-34 converging after 49 iterations), S11 (Lmo2754; IPR001967: E-value ) 1.3 × 10-109), S49 (Lmo1585; IPR004635: E-value ) 8.3 × 10-108), or M23 (Lmo2504; IPR016047: E-value ) 8.1 × 10-51). A number of Sec-translocated proteins lacking cell-wall binding domains encountered in L. monocytogenes were identified as involved in degradation, maturation and/or biogenesis of cell-wall, including the characterized P45 (protein of 45 kDa; Lmo2505) devoid of cell-wall binding domain (Tables 1 and 1S, Supporting Information).87,88 Previous genomic analyses already identified Lmo0394 and Lmo1104 as NlpC/P60 peptidases (IPR000064),89 as well as the N-acetylmuramoyl-L-alanine amidase Lmo1521 (IPR017293).88 However, the cell-wall hydrolase Lmo0019 was identified as such for the first time (PSIBLAST: 1.0 × 10-35 e E-values e1.0 × 10-3 converging at 156 iterations). From previous bioinformatic analyses,16 murami5080

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dases Lmo1215 and Lmo1216 were misleadingly predicted as only localized within the cell wall as the number of GW modules was misconceived. Besides hydrolases, several exoproteins were related to the biogenesis of cell wall. Exoproteins Secreted via Tat Pathway. While two proteins were first predicted as exhibiting a putative Tat-dependent signal peptide,15 this subsequent genomic analysis revealed that Lmo1786 is most certainly a 50S ribosomal protein L35 (IPR001706: E-value ) 1.10 × 10-28). Thus, only one gene was predicted as encoding a Tat protein substrate on the whole genome of L. monocytogenes, that is, a Dyp (dye decolorising peroxidase)-type peroxidase (Lmo0367; IPR006314: E-value ) 2.9 × 10-103) (Tables 1 and 1S, Supporting Information). In Escherichia coli, the homologous protein EfeB (E. coli ferrous iron transporter subunit B), which also exhibits a Tat-dependent signal peptide and is located in the periplasm, was proposed to enable heme iron acquisition without internalization.90 Considering iron acquisition is decisive in successful bacterial infection, this L. monocytogenes Dyp-type peroxidase here uncovered for the first time could constitute a new major virulence factor. Exoproteins Secreted via ABC Transporter. Four bacteriocins (Lmo0335, Lmo0615, Lmo2574, and Lmo2753) were predicted as secreted via an ABC transportation pathway (Tables 1 and 1S, Supporting Information). While Lmo2753 is a

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Figure 3. Features of Sec-dependent signal peptides in exoprotein precusors of L. monocytogenes EGD-e. (A) Length distribution of the complete signal peptides. (B) Length distribution of the n-domains. (C) Length distribution of the h-domains. (D) Frequency of amino acid at position -3 of the cleavage site. (E) Frequency of amino acid at position -2 of the cleavage site. (F) Frequency of amino acid at position -1 of the cleavage site. (G) Frequency of amino acid at position +1 of the cleavage site. Distributions and frequencies are expressed as a percentage of the total number of predicted N-terminal signal peptides in exoproteins. Length is expressed as the number of amino acid residues within the sequence. Amino acid names within the c-domain are given in monoletter code.

leaderless bacteriocin,33 an additional bacteriocin, Lmo2776 homologous to the unusual lactococcin 972 (TIGR01653: Evalue ) 1.0 × 10-83), is most certainly exported in a Secdependent manner.91 Exoproteins Secreted via Holins. The 3 exoproteins were identified as substrates to holins (Tables 1 and 1S, Supporting Information). The autolysin Lmo0129 is predicted as secreted via a TcdE (toxigenic Clostridium difficile protein E)-like holin (Lmo0128) and the endolysin Ply118 (phage lysin of φA118; Lmo2278) secreted via Hol118 (holin of φA118; Lmo2279). The polygalacturonase Lmo2284 (PSI-BLAST: 2.0 × 10-50 e E-values e 1.0 × 10-3 with convergence reached after 323 iterations) exhibiting a pectin/pectate lyase domain (IPR011050: E-values e 5.0 × 10-6), which lacked an N-terminal signal peptide and was encoded in close proximity of hol118, was uncovered as an additional substrate of Hol118 (Tables 1 and 1S, Supporting Information). This exoprotein would thus be involved in degradation of plant cell wall material, which well-matches with the saprophytic lifestyle of L. monocytogenes. Exoproteins Secreted via Wss Pathway. Among exoproteins lacking an N-terminal signal peptide, 3 proteins were predicted as substrates of the Wss (Tables 1 and 1S, Supporting Informa-

tion). Both the paralogues Lmo0056 and Lmo0063 belong to the WXG100 superfamily of secreted proteins.15,92 The protein Lmo0062 homologous to EsaC (ESAT-6 [early secreted antigen target of 6 kDa] secretion accessory protein C) from Staphylococcus aureus was recently reported extracellularly.93 At the opposite to Mycobacterium tuberculosis,94 S. aureus,95 and Bacillus anthracis,96 the Wss of L. monocytogenes was demonstrated as dispensable for bacterial virulence.97 Experimental 2-DE Gels of the Extracellular Proteome from L. monocytogenes EGD-e. This subproteome was subsequently experimentally analyzed by 2-DE. As 95% of proteins predicted as secreted in L. monocytogenes EGD-e have a pI comprised between 3 and 10 (Table 1 and Figure 4), a non linear pH 3-10 IPG strips was used for IEF separation in the first dimension prior to protein resolving according the molecular mass in the second dimension using SDS-PAGE at 12.5% acrylamide. At this single percentage of acrylamide in the resolving gel, a dynamic range of protein separation can be achieved for molecular masses of 10-100 kDa,98 which covers 85% of the exoproteins predicted as secreted in L. monocytogenes EGD-e (Table 1 and Figure 4). As expected from the theoretical 2-DE map, two clusters of proteins could be Journal of Proteome Research • Vol. 9, No. 10, 2010 5081

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Desvaux et al. riocins secreted via ABC exporters could not be experimentally identified as part of exoproteome.

Figure 4. Theoretical 2-DE map of L. monocytogenes EGD-e exoproteome. Following in-depth genomic analysis based on an original and rational strategy mimicking protein secretion process in Gram-positive bacteria, 90 exoproteins were identified as secreted via Sec, Tat, ABC, holins or Wss pathways in L. monocytogenes. Considering cleavable N-terminal signal peptides, a mature set of proteins could be deduced and served for generating this theoretical 2-DE map of the exoproteome. Dotted frame indicates the region that could be observed experimentally in 2-DE using pH 3-10 IPG strips in the first dimension and a SDS-PAGE with 12.5% acrylamide gel in the second dimension. The different colors represent spots corresponding to exoproteins secreted via Sec (yellow), Tat (green), ABC (purple), holin (magenta) and Wss (red) pathways. For theoretical 2-DE maps with detailed labeling of proteins spots, see Figure 1S (Supporting Information).

distinguished experimentally (Figures 3 and 4). The majority of the protein spots were located in a region between pH 4.0 and 6.0, but some strong spots were also present in the basic region. Proteins spots were sampled and further identified by MALDI-TOF MS and peptide mass fingerprinting following interrogation using Mascot. The databank DB-Mature-EGDe previously generated and containing the set of mature proteins as defined from genomic analysis, was first converted to Mascot format and used for identification of protein spots present on 2-DE gel. Peptide mass fingerprinting allowed for a total of 120 spots to be identified, which corresponded to 50 distinct proteins (Table 2). As previously reported, several proteins were resolved as two or more spots on the 2-DE gels.16,19 While most of the 50 proteins were found in single or limited number of spots, 6 proteins were resolved in a higher number of spots (>4), that is, Lmo0202, Lmo0204, Lmo0205, Lmo1786, Lmo1521, and Lmo2591 (Figure 5 and Table 2). Besides post-translational modifications (phoshorylation, glycosylation), reproducible truncations at specific sites would occur for some of these proteins as indicated by mark differences of their theoretical and experimental MM and/or pI (Table 2). Subcellular locations and corrected annotations of these exoproteins experimentally uncovered were performed using the same rational and bioinformatic strategy described above (Table 2S, Supporting Information). Transport of these exoproteins (88%) then appeared as relying essentially on the Sec pathway or via nonclassical secretion. In the experimental conditions used, none of the exoproteins previously predicted as secreted via the Tat, holins or Wss pathway could be identified by proteomics. As expected from the theoretical 2-DE map and because of their low molecular masses (Figure 5), the bacte5082

Journal of Proteome Research • Vol. 9, No. 10, 2010

Predicted Secretion and Subcellular Location of the Proteins Identified in the Supernanant. Most of the proteomically identified proteins (62%) were already considered as localized extracellularly from the genomic analysis previously carried out but some of them were primarily predicted as cytoplasmic, membrane or cell-wall associated (Table 2 and Figure 5). Two proteins (Lmo0130 and Lmo2714) were predicted with a C-terminal LPXTG domain (Table 2S, Supporting Information) and thus would be covalently anchored to bacterial cell wall by sortases.99 Besides Lmo1521 exhibiting only one GW module, 2 additional exoproteins (Lmo2203 and Lmo2591) were identified in the supernantant and predicted with GW modules ranging between 2 and 4 (SSF82057; 2.5 × 10-27 e E-values e 4.2 × 10-12). Three proteins (Lmo0582, Lmo2522, and Lmo2691) were predicted with LysM domains found in 2 or 4 copies (IPR002482: 3.7 × 10-21 e E-values e 2.0 × 10-12), which bind directly to peptidoglycan.100 One protein (Lmo0585) exhibited a WXL domain (Table 2S, Supporting Information) considered as mediating cell-wall association,101 although this has never been experimentally addressed in Listeria.102 All proteins bearing a cell-wall binding domain (LPXTG, GW, LysM, or WXL) were predicted as secreted in a Sec-dependent manner. The situation was the same for lipoproteins (Lmo0013, Lmo0315, Lmo1068, Lmo1388, and Lmo2196), which all exhibited as lipobox located within their N-terminal signal peptide (Table 2S, Supporting Information). Interestingly enough, the 5 lipoproteins experimentally identified as part of the exoproteome of L. monocytogenes EGD-e exhibit a glycine residue at position +2 of the predicted Type II signal peptidase cleavage site (Table 2S, Supporting Information). This situation is considered of major importance in lipoprotein release into the supernatant of Gram-positive bacteria.103-105 Functional Categories of the Identified Exoproteins. All five extracellularly secreted virulence factors so far characterized in L. monocytogenes EGD-e were identified as part of the exoproteome, namely PlcA (Spot 0201), LLO (Spots 0202), PlcB (Spots 0205), MnSOD (Spots 1439) and InlC (Spots 1786), as well as Mpl (Spot 0203) involved in the post-translational maturation of PlcB14 (Table 2 and Figure 5). In addition, the Type I integrated membrane protein ActA (actin assembly), a key virulence factor involved in actin recruitment and motility, was found in the culture supernatant (Spots 0204); such limited release from the bacterial cell surface was previously reported.106 Similarly, a membrane-anchored immunogenic proteins that elicit pathogen-specific CD4+ T-cell responses was also found in the culture supernatant (Spot 1388). In conjunction with exoproteins involved in bacterial pathogenesis, several secreted degradative enzymes most likely related to its saprophytic lifestyle could be identified in L. monocytogenes EGD-e culture supernatants. The noncovalently bound and cell-surface protein CscB (cell-surface complex protein B) (Spot 0585) could be involved in plant carbohydrate utilization but is also considered as a coaggregation-promoting factor.102,107 The chitinase ChiA (Spot 1883) was also identified extracellularly;82 this enzyme is regarded as contributing to chitin colonization and degradation. In addition to enzymes involved in degradation of carbohydrate polymers, an homologue of CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase) was newly identified as part of the exoproteome (Lmo0130; PRK09419: E-value ) 5.0 × 10-49). This enzyme is well-known in Shewanella spp. to play an

research articles

Comprehensive Exoproteome of L. monocytogenes EGD-e

Table 1. Proteins Predicted As Secreted and Localized in the Extracellular Milieu in L. monocytogenes EGD-e Following Genomic Analysis Based on an Original and Rational Strategy Mimicking Protein Secretion Process in Gram-Positive Bacteria protein name

secretion systema

annotationb

MM (KDa)c

pIc

Lmo0201 Lmo0202 Lmo0203 Lmo0205 Lmo1439 Lmo1786 Lmo0367

Sec Sec Sec Sec Sec Sec Tat

Bacterial virulence Phosphatidylinositol phospholipase C, PlcA Listeriolysin O (Thiol-activated cytolysin) (LLO) Hly (HlyA) (LisA) Zinc metalloproteinase, Mpl (PrtA) Phosphatidylcholine phospholipase C, PlcB (PrtC) Manganese-superoxide dismutase, MnSOD Internalin C, InlC Dyp-type peroxidase

32.9 56.0 54.7 30.6 22.6 30.7 42.4

9.6 7.2 6.2 6.9 5.2 5.7 5.8

Lmo0105 Lmo0507 Lmo0540 Lmo0755 Lmo1128 Lmo1264 Lmo1333 Lmo1499 Lmo1511 Lmo1585 Lmo1654 Lmo1862 Lmo1883 Lmo1913 Lmo2056 Lmo2074 Lmo2444 Lmo2467 Lmo2504 Lmo2754 Lmo2284

Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Holin

Degradative enzymes Chitinase, ChiB Phosphotransferase system, galactitol-specific IIB component β-lactamase-type transpeptidase SGNH hydrolase-type esterase Lysophospholipase Metalloprotease with zincin-like fold Aminodeoxychorismate lyase Aminodeoxychorismate lyase Lysophospholipase Peptidase S49 Metalloprotease with zincin-like fold SGNH hydrolase-type esterase Chitinase, ChiA Six-hairpin glycosidase-like Ca2+ chelating serine protease with SCP/PR1 domain Peptidase S9 Glycosyl hydrolase family 31 with galactose-binding like domains Chitin binding protein Peptidase M23 Peptidase S11, D-alanyl-D-alanine carboxypeptidase A Polygalacturonase

78.2 7.7 38.9 26.9 33.8 16.7 14.3 37.1 22.8 33.5 15.2 26.8 35.2 35.3 37.0 32.0 140.4 49.3 44.5 44.9 37.2

5.2 4.6 8.4 5.2 8.9 9.9 8.0 6.3 5.2 4.5 9.5 5.3 5.1 5.3 5.8 5.6 4.3 5.4 5.7 5.8 5.2

Lmo0019 Lmo0394 Lmo0415 Lmo0441 Lmo0971 Lmo1104 Lmo1215 Lmo1216 Lmo1438 Lmo1521 Lmo1547 Lmo2039 Lmo2505

Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec Sec

27.0 21.4 49.3 69.5 45.0 33.8 29.2 34.2 74.0 43.0 28.2 77.4 39.9

10.4 10.0 9.3 5.6 8.7 4.6 5.3 6.3 5.3 5.3 4.9 6.0 7.8

Lmo2713 Lmo0129 Lmo2278

Sec Holin Holin

Cell wall degradation and biogenesis Cell wall hydrolase with SH3 domain NlpC/P60-type cell wall hydrolase Peptidoglycan GlcNAc deacetylase Cell division protein FtsI/Penicillin-binding protein 2, transpeptidase D-alanine esterification of lipoteichoic acid and wall teichoic acid protein, DltD NlpC/P60-type cell wall hydrolase Muramidase flagellum-specific with a single GW domain, FlgJ-type Muramidase flagellum-specific with a single GW domain, FlgJ-type Cell division protein FtsI/Penicillin-binding protein 2, transpeptidase N-acetylmuramoyl-L-alanine amidase with a single GW domain, YrvJ-type Cell shape-determining protein, MreC Cell division protein FtsI/Penicillin-binding protein 2, transpeptidase Peptidoglycan lytic protein P45 (protein of 45 kDa), Spl (secreted protein with lytic property) YkuD family protein (former ErfK/YbiS/YcfS/YnhG family protein) Cell wall hydrolase, autolysin Endolysin Ply118

32.3 26.5 30.8

9.9 9.0 9.9

Lmo0193 Lmo0275

Sec Sec

Antibiotic resistance Macrolide transporter subunit MacA Metallo-β-lactamase

21.1 27.1

5.0 10.1

Lmo0017

Sec

37.9

5.3

Lmo0516

Sec

Polyglutamate biosynthesis Poly-γ-glutamate biosynthesis enzyme, 5′-nucleotidase/2′,3′-cyclic phosphodiesterase, UDP-sugar hydrolase Poly-γ-glutamate biosynthesis enzyme

50.9

5.7

Lmo1966 Lmo2106

Sec Sec

Phosphatases 5-bromo-4-chloroindolyl phosphate hydrolysis protein Metallo-dependent phosphatase

20.9 28.4

9.5 5.8

Lmo1484

Sec

Competence DNA uptake competence protein, ComEA

17.7

5.0

Sec

Signal transduction Signal transduction YycH protein

46.7

4.8

212.6 82.7 8.4 25.0 19.5 15.1 14.3 36.0 18.5

4.9 5.3 4.4 4.8 8.9 4.7 4.4 4.7 4.6

Lmo0289 Lmo0086 Lmo0087 Lmo0206 Lmo0412 Lmo0438 Lmo0461 Lmo0462 Lmo0601 Lmo0671

Sec Sec Sec Sec Sec Sec Sec Sec Sec

Sec-translocated Sec-translocated Sec-translocated Sec-translocated Sec-translocated Sec-translocated Sec-translocated Sec-translocated Sec-translocated

Unspecific or unknown function protein of unknown function with fibronectin type III-like fold protein of unknown function protein of unknown function protein of unknown function, COG3109 protein of unknown function protein of unknown function protein of unknown function protein of unknown function with WD-40 repeat, COG3595 protein of unknown function

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Table 1. Continued protein name

secretion systema

Lmo0724 Lmo0745 Lmo0778 Lmo0881 Lmo0950

Sec Sec Sec Sec Sec

Lmo0951

Sec

Lmo1601

Sec

Lmo1656 Lmo1752 Lmo2027

Sec Sec Sec

Lmo2093 Lmo2119 Lmo2156 Lmo2217 Lmo2439 Lmo2470

Sec Sec Sec Sec Sec Sec

Lmo2568 Lmo2639 Lmo2686 Lmo2710 Lmo2809

Sec Sec Sec Sec Sec

Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown DUF1801 domain, COG4814 Sec-translocated protein of unknown DUF1801 domain, COG4814 Sec-translocated protein of unknown domain, COG4980 Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown protein, COG4886 Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown protein, COG4886 Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown Sec-translocated protein of unknown

Lmo0335 Lmo0615 Lmo2574 Lmo2753 Lmo2776

ABC ABC ABC ABC Sec

Bacteriocin, class II microcin Bacteriocin Bacteriocin Bacteriocin leaderless Bacteriocin, lactococcin 972

Lmo0056 Lmo0062 Lmo0063

Wss Wss Wss

WXG100-A, Lmesat6 EsaC WXG100-B

annotationb

MM (KDa)c

pIc

function UCP032442 type, COG4990 function function function function with R/β-hydrolase fold and

23.4 15.0 10.2 14.5 28.9

9.1 4.6 4.7 5.5 5.5

function with R/β-hydrolase fold and

27.0

7.3

function with TMP (tape measure protein)

15.0

7.9

function function function with leucine-rich repeat (LRR)

12.5 24.9 37.4

10.0 4.9 3.7

function function YbbR-like, COG4856 function YxeA-like with DUF1093 domain function, COG4980 function YxeA-like, COG5294 function with leucine-rich repeat (LRR)

4.1 45.6 11.1 10.7 9.6 39.7

10.8 5.0 9.4 5.3 5.9 3.9

function YxeA-like, COG5294 function with DUF1312 domain, COG5341 function function function with DUF1310 domain

8.9 11.5 16.8 21.0 11.1

9.6 4.9 8.4 4.6 5.0

7.9 5.8 7.7 9.2 12.0

9.4 4.4 9.4 9.8 10.1

11.4 15.5 11.2

4.6 9.1 9.2

Bacteriocins

Wss secreted proteins

a Protein secretion systems: Sec (Secretion), Tat (Twin-arginine translocation), ABC (ATP-binding cassette), holin (hole forming) and Wss (WXG100 secretion system) pathways. b Some annotations were corrected respective to the biofinformatic analyses performed and details in Table 1S (Supporting Information). For proteins of unknown function, COG was provided whenever possible. c Theoretical average molecular mass (MM) and pI are given for the predicted mature exoprotein, that is, devoid of signal peptide.

important role in the extracellular degradation of DNA, which then serves as a nutrient source of phosphorus, carbon and energy.108 Other enzymes could be involved in degradation of lipids, proteins and/or peptides degradation, such as Lmo1068 (Spots 1068) belonging to GDSL family of esterases/lipases (PSIBLAST: 3.0 × 10-37 e E-values e 2.0 × 10-21 with convergence reached after 73 iterations)109 or Lmo2504 a homologue to a peptidase of the M23 family (IPR011055: E-value ) 1.0 × 10-54), that is, metallopeptidases belonging to MEROPS peptidase family clan M.110 Some proteins primarily involved in cell wall degradation and/or maturation were present extracellularly. This is most certainly associated with their catalytic activity and/or cellwall turnover resulting in their release from bacterial cell surface, which could further explain the presence in the extracellular milieu of proteins exhibiting LPXTG, GW, LysM or WXL motifs primarily predicted as cell-wall associated. Among the cell-wall hydrolases experimentally characterized in L. monocytogenes,87,88 P45 (Spots 2505) and Iap (Spots 0582) could be here experimentally identified as part of the exoproteome. In addition, recently uncovered cell-wall hydrolases Lmo1521 (Spot 1521) and Lmo2522 (Spot 2522) were experimentally identified,19 together with muramidases Lmo2203 (Spots 2203) and Lmo2591 (Spots 2591) specifically involved in flagellum assembly (COG1705: E-values e 3.0 × 10-51) and a murein transglycosylase Lmo0186 (Spot 0186) 5084

Journal of Proteome Research • Vol. 9, No. 10, 2010

involved in recycling of muropeptides during cell elongation and/or division (IPR010611: E-value ) 1.9 × 10-33; COG2821: E-value ) 2.00 × 10-4). Besides enzymes engaged in hydrolysis of murein, several proteins were related to peptidoglycan biosynthesis for cell wall formation in the course of cell division, namely two penicillin-binding proteins FtsI (Lmo1438 and Lmo2039; COG0768: E-values e 1.0 × 10-108) as well as two cell envelope-related transcriptional attenuators (Lmo0443 and Lmo2518; IPR004474: E-values e 3.0 × 10-66; Spots 1438, 2039, 0443, and 2518, respectively). Overall, release of proteins from the cell envelope, which interface the bacterial cell and its surroundings, is taking place in L. monocytogenes EGD-e and further indicates it is the siege of a high activity, which can have numerous consequences on cellular processes including protein secretion, cell growth, biofilm formation, genetic competence, and/or pathogenicity.88 Several proteins primarily predicted as located within the cytoplasm were experimentally identified as part of the exoproteome of L. monocytogenes EGD-e; no N-terminal signal peptide could be identified, no protein secretion system could be attributed and no obvious biological function in the extracellular milieu could be proposed for any of them. Nonetheless, their potential extracellular localization was already predicted from the genomic analysis carried out as they were part of the 215 proteins predicted as released

research articles

Comprehensive Exoproteome of L. monocytogenes EGD-e

Table 2. Exoproteome of L. monocytogenes EGD-e Following Protein Spots Identification by MALDI-TOF MS from 2-DE Gel protein IDa

annotation

b

spot IDc scored E-valuee

theoreticalf experimental pI

MM

pI

queries queries sequence subcellular secretion MM matched searched coverage locationg systemh

Lmo0201 Phosphatidylinositol phospholipase C, PlcA 0201a 0202a Lmo0202 Listeriolysin O (Thiol-activated cytolysin) (LLO) Hly (HlyA) (LisA) 0202b 0202c 0202d 0202e 0202f 0202g Lmo0203 Zinc metalloproteinase, Mpl (PrtA) 0203a Lmo0204 ActA Actin-assembly inducing protein 0204a 0204b 0204c 0204d 0204e 0204f 0204g 0204h 0205a Lmo0205 Phosphatidylcholine phospholipase C, PlcB (PrtC) 0205b 0205c 0205d 0205e 0205f 0205g 0205h 0205i 0205j Lmo0582 Iap invasion associated protein (P60) 0582a 0582b 0582c + 1388a Lmo1388 CD4 T-cell stimulating antigen 1439a Lmo1439 Manganese-superoxide dismutase, MnSOD 1439b Lmo1786 Internalin C, InlC 1786a 1786b 1786c 1786d 1786e 1786f 1786g

155 238 215 196 169 164 92 98 52 166 128 91 71 61 56 52 48 188 143 128 115 98 96 94 92 79 58 167 60 59 111 124 50 176 162 116 82 76 62 56

Bacterial virulence 9.0 × 10-13 9.6 32.9 4.5 × 10-21 7.2 56.0 9.0 × 10-19 7.2 56.0 7.1 × 10-17 7.2 56.0 3.6 × 10-14 7.2 56.0 1.1 × 10-13 7.2 56.0 1.6 × 10-6 7.2 56.0 4.9 × 10-7 7.2 56.0 1.9 × 10-2 6.2 54.7 7.1 × 10-14 5.0 70.3 4.5 × 10-10 5.0 70.3 2.2 × 10-6 5.0 70.3 2.1 × 10-4 5.0 70.3 2.1 × 10-3 5.0 70.3 1.0 × 10-2 5.0 70.3 1.6 × 10-2 5.0 70.3 4.1 × 10-2 5.0 70.3 4.5 × 10-16 6.9 30.6 1.4 × 10-11 6.9 30.6 4.5 × 10-10 6.9 30.6 9.0 × 10-9 6.9 30.6 5.1 × 10-7 6.9 30.6 7.1 × 10-7 6.9 30.6 1.0 × 10-6 6.9 30.6 1.8 × 10-6 6.9 30.6 3.6 × 10-5 6.9 30.6 4.3 × 10-3 6.9 30.6 5.7 × 10-14 9.2 48.0 2.5 × 10-3 9.2 48.0 3.9 × 10-3 9.2 48.0 2.3 × 10-8 4.8 36.1 1.1 × 10-9 5.2 22.6 2.5 × 10-2 5.2 22.6 7.1 × 10-15 5.7 30.7 1.8 × 10-13 5.7 30.7 7.1 × 10-9 5.7 30.7 2.0 × 10-5 5.7 30.7 8.0 × 10-5 5.7 30.7 1.9 × 10-3 5.7 30.7 7.0 × 10-3 5.7 30.7

9.9 6.6 6.8 7.2 7.2 6.9 6.8 7.6 6.6 4.7 4.8 4.4 4.7 7.0 4.7 4.9 4.7 6.9 7.0 7.0 7.0 7.0 7.8 7.4 7.0 7.7 7.0 9.3 9.2 9.1 4.6 5.0 4.8 6.1 5.0 5.6 5.1 5.0 4.9 5.6

32.1 57.4 57.4 60.7 60.7 60.7 48.1 46.7 18.8 70.3 69.1 60.2 35.1 27.3 22.6 55.8 35.1 27.0 27.0 24.7 24.6 25.7 22.6 22.6 23.2 24.7 27.1 53.0 35.3 34.8 35.8 22.6 22.6 24.2 24.5 28.5 28.2 28.0 25.1 24.4

15 22 23 19 21 18 11 14 6 17 18 11 11 10 7 10 6 15 13 14 10 13 12 13 11 9 5 15 8 9 12 11 6 13 15 13 10 8 7 8

40 34 47 31 69 37 36 52 21 35 65 32 50 50 31 58 26 22 22 36 30 51 39 52 31 50 16 30 50 50 35 32 45 25 38 48 54 34 34 87

48 47 51 41 46 42 31 34 13 34 35 19 19 23 11 18 11 55 35 42 42 47 37 45 32 40 28 44 21 26 37 52 35 47 54 50 41 33 28 33

EC EC

Sec Sec

EC MB

Sec YidC

EC

Sec

CW

Sec

MB EC/CP

Sec Sec

EC

Sec

Lmo0130 Bifunctional 2′,3′-cyclic nucleotide 2′phosphodiesterase/3′-nucleotidase, CpdB Lmo0540 β-lactamase-type transpeptidase Lmo0585 CscB cell-surface complex protein B Lmo1068 GDSL-like lipase/acylhydrolase

151 108 83 52 66 56 103 83 59 78 68 49

Degradative enzymes 2.3 × 10-12 4.7 76.2 4.5 × 10-8 4.7 76.2 1.3 × 10-5 8.4 38.9 1.7 × 10-2 4.4 21.7 6.5 × 10-4 5.4 28.7 6.7 × 10-3 5.4 28.7 1.4 × 10-7 8.0 14.3 1.3 × 10-5 8.0 14.3 3.8 × 10-3 5.1 35.2 4.2 × 10-5 5.7 44.5 4.8 × 10-4 5.7 44.5 3.7 × 10-2 5.7 44.5

4.6 4.6 6.8 9.1 5.6 5.7 7.8 9.6 4.9 5.5 5.6 5.5

73.0 74.0 35.6 34.8 30.1 28.2 13.6 12.0 35.4 51.0 77.9 77.9

20 16 10 6 9 7 9 9 8 9 10 7

50 50 43 31 67 49 31 50 50 26 50 47

41 30 34 42 42 36 59 60 33 25 35 18

CW

Sec

EC CW MB

Sec Sec Sec

EC

Sec

EC EC

Sec Sec

degradation and biogenesis 2.8 × 10-7 9.0 44.5 9.0

44.4

10

29

28

MB

YidC

1.4 × 10-11 7.1 × 10-8 1.5 × 10-5 5.7 × 10-13 1.8 × 10-12 9.0 × 10-18 1.1 × 10-16 7.1 × 10-16 2.3 × 10-15 5.7 × 10-13 5.1 × 10-3 1.6 × 10-2

9.1 9.1 9.1 5.3 5.3 5.3 5.3 5.3 5.3 5.3 5.3 6.0

34.1 34.1 34.1 74.0 74.0 43.0 43.0 43.0 43.0 43.0 43.0 77.4

5.5 5.1 4.9 5.0 5.1 5.2 5.4 4.9 5.0 4.8 5.4 5.4

22.6 22.6 22.6 68.8 68.8 47.4 27.4 47.4 47.4 47.4 47.6 69.6

13 13 10 23 23 19 17 16 17 14 7 10

30 44 45 60 71 43 35 32 40 31 30 50

45 36 36 36 39 57 53 50 53 47 22 19

MB

YidC

EC

Sec

EC

Sec

EC

Sec

-16

5.0 5.0 7.8 7.8 7.8 7.8 5.6 5.6 5.3 5.3 5.3 5.8 5.8

37.8 37.8 39.9 39.9 39.9 39.9 39.1 39.1 26.8 26.8 26.8 54.2 54.2

4.7 4.9 7.2 7.8 6.7 6.2 4.6 4.6 5.0 4.9 4.8 5.8 5.5

35.1 35.1 44.0 44.0 44.0 45.0 33.0 32.2 26.5 26.0 27.8 57.0 56.7

14 9 16 16 13 7 9 9 10 11 8 28 27

24 51 28 29 45 42 33 40 29 68 24 39 55

52 32 44 44 37 26 25 24 54 60 42 58 53

CW

Sec

EC

Sec

Lmo1333 Aminodeoxychorismate lyase Lmo1883 Chitinase, ChiA Lmo2504 Peptidase M23

0130a 0130b 0540a 0585a 1068a 1068b 1333a 1333b 1883a 2504a 2504b 2504c

Cell-wall Lmo0186 Membrane-bound lytic murein 0186a 100 transglycosylase with 3D, G5 and DUF348 domains Lmo0443 Cell envelope-related transcriptional 0443a 143 attenuator 0443b 106 0443c 83 1438a 157 Lmo1438 Cell division protein FtsI/Penicillinbinding protein 2, transpeptidase 1438b 152 1521a 205 Lmo1521 N-acetylmuramoyl-L-alanine amidase with a single GW domain, YrvJ-type 1521b 194 1521c 186 1521d 181 1521e 157 1521f 58 52 Lmo2039 Cell division protein FtsI/Penicillin-binding 2039a protein 2, transpeptidase Lmo2203 Muramidase flagellum-specific 2203a 185 2203b 65 2505a 201 Lmo2505 Peptidoglycan lytic protein P45 (protein of 45 kDa), Spl (secreted 2505b 198 protein with lytic property) 2505c 117 2505d 50 Lmo2518 Cell envelope-related transcriptional 2518a 85 attenuator 2518b 62 Lmo2522 Cell-wall associated autolysin with 3D 2522a 99 domain 2522b 78 2522c 70 Lmo2591 Muramidase flagellum-specific with 2591a 341 GW domains 2591b 263

9.0 × 10 9.4 × 10-4 2.3 × 10-17 4.5 × 10-17 5.7 × 10-9 2.7 × 10-2 8.6 × 10-6 1.8 × 10-3 3.7 × 10-7 4.6 × 10-5 2.9 × 10-4 2.3 × 10-31 1.4 × 10-23

MB

YidC

CW

Sec

CW

Sec

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Table 2. Continued protein IDa

annotationb

Lmo2691 Cell-wall associated autolysin

Lmo0013 QoxA AA3-600 quinol oxidase subunit II Lmo0135 Bacterial extracellular solute-binding protein of ABC-type dipeptide transport system, family 5 Lmo2196 ABC-type oligopeptide transport system, solute-binding protein component Lmo0196 Stage V sporulation protein G Lmo0211 50S ribosomal protein L25/General stress protein Ctc/Gln-tRNA synthetase (GlnRS), anticodon-binding domain Lmo0223 Cysteine synthase A Lmo0796 Lipid/polyisoprenoid-binding, YceI-like Lmo1055 Dihydrolipoamide dehydrogenase Lmo1364 Cold shock protein, CspA type Lmo1473 DnaK class I heat-shock chaperone protein Lmo2068 Chaperonin GroEL Lmo2069 Chaperonin GroES Lmo2455 Enolase Lmo2456 Phosphoglycerate mutase, 2,3-bisphosphoglycerateindependent Lmo2458 Phosphoglycerate kinase Lmo2459 Glyceraldehyde-3-phosphate dehydrogenase Lmo2511 Ribosomal protein S30Ae/σ54 modulation protein Lmo0412 Sec-translocated protein of unknown function, COG3109 Lmo0644 Membrane-bound sulphatase, HI1246 type, COG1368 Lmo0927 Membrane-bound sulphatase, HI1246 type, COG1368 Lmo1752 Sec-translocated protein of unknown function Lmo2156 Sec-translocated protein of unknown function YxeA-like with conserved DUF1093 domain Lmo2714 Protein of unknown function with LPXTG motif

spot IDc scored E-valuee

theoreticalf experimental pI

MM

pI

queries queries sequence subcellular secretion MM matched searched coverage locationg systemh

5.8 5.8 5.8 5.8 9.7 9.7

54.2 54.2 54.2 54.2 57.9 57.9

6.3 5.3 5.0 5.0 4.5 6.1

56.3 56.9 56.5 56.5 15.3 38.0

23 14 13 12 8 7

54 23 31 35 21 15

51 29 31 26 13 12

Membrane transport 3.9 × 10-3 5.7 39.4 1.3 × 10-2 5.7 39.4 3.3 × 10-2 5.7 39.4 5.7 × 10-9 5.0 56.2

5.0 5.1 5.2 4.7

33.2 33.2 41.8 55.7

10 8 8 16

50 45 48 55

4.8

63.5

11

Exported by nonclassical secretion 0196a 80 3.1 × 10-5 4.5 11.3 4.5 0211a 61 2.1 × 10-3 4.4 22.7 4.5

11.2 22.6

2591c 2591d 2591e 2591f 2691a 2691b

220 163 127 104 73 72

0013a 0013b 0013c 0135a

59 53 49 117

2196a

73

2.8 × 10-19 1.4 × 10-13 5.7 × 10-10 1.1 × 10-7 1.5 × 10-4 1.8 × 10-4

1.4 × 10-4

5.1

60.2

CW

Sec

32 30 26 42

MB

Sec

MB

Sec

52

29

MB

Sec

9 5

50 17

56 31

EC/CP EC/CP

NC NC

0223a 0796a 1055a 1364a 1473a 1473b 1473c 1473d 2068a 2068b 2069a 2455a 2456a

108 49 50 50 157 55 48 48 161 161 105 233 52

4.5 × 10-8 3.4 × 10-2 2.8 × 10-2 3.1 × 10-2 5.7 × 10-13 9.9 × 10-3 4.7 × 10-2 4.7 × 10-2 2.3 × 10-13 2.3 × 10-13 9.0 × 10-8 1.4 × 10-20 1.9 × 10-2

5.3 4.7 5.2 4.5 4.6 4.6 4.6 4.6 4.7 4.7 4.6 4.7 5.1

32.2 19.3 49.5 7.3 66.1 66.1 66.1 66.1 57.4 57.4 10.1 46.5 56.1

5.0 4.5 5.0 4.5 4.5 4.5 4.6 4.6 4.6 4.6 4.6 4.5 4.5

32.2 15.3 56.5 9.5 68.0 66.0 71.0 66.5 60.0 69.0 10.0 47.1 15.3

13 5 7 4 20 9 9 9 20 20 8 22 7

45 50 40 48 50 50 50 50 50 50 26 39 50

52 34 17 74 43 24 16 16 39 39 82 59 19

EC/CP EC/CP EC/CP EC/CP EC/CP

NC NC NC NC NC

EC/CP

NC

EC/CP EC/CP EC/CP

NC NC NC

2458a 2459a 2459b 2459c 2511a

57 139 139 77 60

5.8 × 10-3 3.6 × 10-11 3.6 × 10-11 5.8 × 10-5 2.9 × 10-3

5.0 5.2 5.2 5.2 5.3

42.1 36.3 36.3 36.3 21.6

4.8 4.9 5.0 5.0 5.1

45.8 36.5 36.9 36.2 19.9

8 13 13 12 5

52 30 30 55 24

34 42 42 32 49

EC/CP EC/CP

NC NC

EC/CP

NC

Unspecific or unknown function 71 2.4 × 10-04 4.8 25.0 4.6

26.8

9

49

43

EC

Sec

0412a

-13

0644a 0644b 0927a 0927b 0927c 0927d 1752a 1752b 2156a

164 160 174 143 56 50 137 55 61

1.1 × 10 2.8 × 10-13 1.1 × 10-14 1.4 × 10-11 6.7 × 10-3 3.0 × 10-2 5.7 × 10-11 8.4 × 10-3 2.3 × 10-3

5.4 5.4 6.0 6.0 6.0 6.0 4.9 4.9 9.4

69.3 69.3 74.7 74.7 74.7 74.7 24.9 24.9 11.1

4.6 4.7 4.8 4.8 4.9 4.8 4.7 4.8 10.0

48.0 48.0 53.0 45.8 52.6 53.0 32.0 30.0 9.9

24 21 18 16 12 8 10 7 6

67 60 35 36 65 30 21 35 28

41 39 32 30 16 8 37 31 46

MB

YidC

MB

YidC

EC

Sec

EC

Sec

2714a

61

2.3 × 10-3

4.6

28.7

4.6

33.0

9

59

45

CW

Sec

a As highlighted by underlined protein names and as indicated in the column entitled “Subcellullar location”, some of these experimentally identified exoproteins were already predicted as secreted by known protein secretion systems and located in the extracellular milieu (EC) by genomic analyses (Table 1). b Some annotations were corrected respective to the bioinformatic analyses performed and detailed in Table 2S. For proteins with unspecific or unknown function, COG was provided whenever possible. c The first four digits used as spot identifier (ID) corresponds to the identification number for protein in L. monocytogenes EGD-e, for example the spot “0201a” corresponds to protein Lmo0201. d Mascot score was obtained against the databank DB-Mature-LmoEGDe v1.0 previously generated and containing the set of mature exoproteins of L. monocytogenes EGD-e as defined from genomic analysis, where scores greater than 47 are significant (p < 0.05). e E-value was associated with Mascot score established with a significant threshold set up at p < 0.05 and obtained against DB-Mature-LmoEGDe v1.0. f Theoretical average pI and molecular mass (MM) are given for the predicted mature exoprotein, that is, devoid of signal peptide. g Predicted protein subcellular location: EC, extracellular milieu; CW, cell wall; MB, membrane; and CP, cytoplasm. Detailed bioinformatic analysis are provided in Table 2S, Supporting Information. h Predicted protein secretion system involved: Sec (Secretion) pathway, YidC insertase and nonclassical (NC) protein secretion systems. Integration of membrane proteins could occur via YidC a Sec-dependent or independent manner.120 Detailed bioinformatic analysis are provided in Table 2S, Supporting Information.

via nonclassical secretion. Some of these proteins are first and foremost involved in central metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or enolase (Spots 2455 and 2459 respectively).84,111 Clearly, these proteins can have multiple final subcellular locations and have been demonstrated to moonlight on the cell surface of L. monocytogenes by binding human plasminogen.16,68 5086

Journal of Proteome Research • Vol. 9, No. 10, 2010

This proteomic analysis also revealed the expression of several proteins whose genes were originally annotated as hypothetical and for which no further insight in their putative function could be gained from additional in silico analyses; 4 of them (Lmo0412, Lmo1752, Lmo2156, and Lmo2714) exhibited a Sec-dependent signal peptide, including one LPXTG protein, Lmo2714.

Comprehensive Exoproteome of L. monocytogenes EGD-e

research articles

Figure 5. Empirical 2-DE map of L. monocytogenes EGD-e exoproteome. Proteins from the extracellular fraction were run in the first dimension by IEF in nonlinar pH 3-10 IPG strips and in SDS-PAGE 12.5% acrylamide gel in the second dimension. The first four digits used for spot labeling corresponds to the identification number for gene/protein in L. monocytogenes EGD-e, for example spots labeled as “0201” corresponds to protein Lmo0201. For experimental 2-DE maps with detailed labeling of protein spots, see Figure 2S (Supporting Information).

Discussion Besides mimicking the molecular events encountered by a protein in the course of secretion across the cell envelope of a monoderm bacterium, the core originality of the prediction strategy applied resides in taking into account protein secretion systems, which are the gating systems enabling protein translocation across a biological membrane and consequently the key event in any subcellular location process. In addition to the Sec system, the protein secretion via Tat, FPE, and ABC pathways was also regarded for proteins exhibiting a N-terminal signal peptide. This is in mark contrast with previous genomic approaches used to predict exoproteome of L. monocytogenes.16,17 Consideration of the systems gating the translocation of proteins across the cytoplasm is certainly one of the major advances for the correct prediction of extracellular proteins, which allowed us to strongly refining the prediction of exoproteins in L. monocytogenes EGD-e. By taking into account both the secretion systems and the secreted proteins, this investigation of the exoproteome truly corresponds to a secretomic analysis. Compared to previous genomic analyses of the exoproteome of L. monocytogenes EGD-e16,17 and available databases dedicated to protein subcellular location in genome-sequenced Gram-positive bacteria,112-115 our rational approach mimicking the sequence of events occurring in a bacterial cell in the course

of protein secretion greatly improved the prediction of exoproteins (Table 3S, Supporting Information). More than quantitatively, the present investigation allowed to qualitatively improving the prediction of exoproteins in L. monocytogenes. For example, prepseudopilins translocated via the FPE were not reported as exoproteins, prepeptides extracellularly secreted via dedicated ABC exporters were considered or GW proteins exhibiting only one module were described as released in the extracellular milieu. The bioinformatic strategy we described represents a major stepforward in genomics and proteomics in the field of protein secretion. It lacks the rigidity of SVMs or pipelines available online and gains in flexibility as new predictive tools can be implemented as soon as they become available. This approach is generic and has been purposely designed for the prediction of proteins localized extracellularly in other monoderm bacteria. Besides genomic analysis, this rational strategy is also useful for categorizing, analyzing, and supporting proteomic data related to proteins identified in culture supernatant. From our genomic analysis, a database containing mature protein sequences could be generated. This is particularly pertinent and rigorous for proteomic analysis focusing on the exoproteome. Indeed, standard database provided by GenBank and further converted to Mascot format for peptide mass fingerprinting, systematically contains proteins with their NJournal of Proteome Research • Vol. 9, No. 10, 2010 5087

research articles terminal signal peptides, which are not present in maturated exoproteins as it is cleaved in the course of secretion. The use of such standard databases is not really appropriate as it can lead to biased scores, and ultimately to invalidating scores, especially for small proteins, even so it might correspond to the right protein for a given spot; this issue remains true for nongel proteomic approaches as protein identification is also based on expected peptide sizes. Besides significant differences for theoretical pI and MM, scores and E-values for protein identification were systematically better for protein exhibiting a predicted signal peptide when using the mature database (DB-Mature-LmoEGDe v1.0) compared to a database provided by GenBank (Table 4S, Supporting Information); 5 spots and 2 proteins would not have been identified using a standard database. In addition, the mature database allowed confirming experimentally the postulated cleavage site of the signal peptide in several exoproteins, for example, the virulence factors PlcA (Lmo0201) or Iap (Lmo0582) (Table 4S, Supporting Information). As most research works performed on L. monocytogenes, our investigations focused on conditions related to host colonization and infection associated with the expression of virulence factors, namely by growing bacteria at 37 °C in chemically defined medium. However, these experimental conditions are quite human centered but are certainly radically different from the environmental conditions what are expected to prevail in the natural environment of this bacterium, whether in soil, food and other natural products. It would indeed be of great interest to investigate how various environmental conditions alter the pattern of the exoproteome. For example, the regulation of genetic expression in function of the temperature is well-known and particularly relevant in L. monocytogenes.116 Besides differential protein expression in various growth conditions, such investigations could lead to the experimental identification of new exoproteins as predicted by our genomic analysis. This investigation resulted in the really first comprehensive appraisal of the exoproteome of L. monocytogenes EGD-e based on theoretical and experimental 2-DE maps, which further provided indications on listerial physiology in relation with its habitat and lifestyle. The 50 identified exoproteins were essentially virulence factors, degradative enzymes and proteins of unknown functions. Out of them, 31 were originally predicted as secreted into the extracellular milieu, including 17 in a Sec-dependent manner and 14 via nonclassical secretion. However, among the remaining 19 experimentally identified exoproteins, 13 were predicted as Sec secreted but located in the cell envelope. Five of them were lipoproteins, which systematically exhibited a glycine residue at position +2 respective to the cleavage site; as suggested in other Grampositive bacteria, this could constitute a signal exclusion leading to the release of lipoproteins from the membrane.103-105 The remaining experimentally identified Sec-secreted exoproteins were originally predicted as located in the cell wall. As suggested by the presence of secreted enzymes involved in the degradation, maturation and biogenesis of the cell wall, the presence of proteins exhibiting cell-wall binding motifs could be collateral.88 Alternatively, it cannot be excluded that for some of these experimentally identified exoproteins the discrepancy between their presence in the culture supernatant and their predicted location results from mispredictions. Nonetheless, protein secretion would rely essentially on the Sec pathway in L. monocytogenes, that is, 30 proteins exhibiting a Sec-dependent signal peptide. With regard to the number of 5088

Journal of Proteome Research • Vol. 9, No. 10, 2010

Desvaux et al. predicted Sec-secreted exoproteins, only a fraction could be experimentally identified, that is, 17 out of 79 predicted proteins. As a matter of fact, only Sec-secreted proteins could be experimentally identified among exoproteins predicted as exported via known secretion pathways; no protein transported via Tat, ABC, holin or Wss could be here uncovered. Besides regulation of gene expression under different environmental conditions as mentioned above, this discrepancy with the predicted exoproteome could also result from technical limitations such as pI/MM ranges in 2-DE, low level of protein expression combined with insufficient staining intensity or the sensitivity of MS apparatus. Anyhow, considering prediction on their limited prevalence over Sec-secreted exoproteins, the identification of proteins secreted via alternative pathways in L. monocytogenes would necessitate more focus molecular analysis to ensure for example that protein secretion system and the substrate are at least expressed at transcriptomic level, in which environmental conditions and if they are indeed functional. Similarly, except for secreted virulence factors, biological functions of most expressed exoproteins remain putative and a large part of the proteins we identified have unspecific or unknown function. Several proteins primarily predicted as cytoplasmic were experimentally identified as part of the exoproteome in L. monocytogenes. All cytoplasmic proteins identified in the extracellular milieu were actually predicted to follow nonclassical secretion. Some proteins might actually be secreted in a Sec-dependent manner but in conjunction with SecA2 instead of SecA,15 as reported for primarily cytoplasmic proteins GroEL117 and MnSOD81 both lacking a putative N-terminal signal peptide. While extensive cell lysis in early stationary phase could be ruled out by assaying the strictly cytoplasmic aminopeptidase C (no activity detected in the supernatant), contribution of tightly controlled cell lysis such as allolysis rather than autolysis cannot be completely excluded.118 Besides cell lysis, several alternative hypotheses have been formulated to explain the extracellular presence of these primarily cytoplasmic proteins such as uncovered secretion systems, piggybacking or leakage. Unveiling the molecular mechanisms responsible for the presence of each of these proteins in the extracellular milieu undoubtedly requires further in-depth investigations in Gram-positive bacteria. Altogether, it appears evident that much remained to be learned about the physiology of L. monocytogenes in a context broader than the sole infection cycle. A clear feature of this species, sometimes overlooked by researchers mind-focused on bacterial virulence, is that L. monocytogenes is primarily a ubiquitous free-living saprophyte that can come in contact with a human host, where it can sometimes induce diseases but rarely have lethal outcome except in special circumstances.119 Characterization of molecular mechanisms related to the adaptation and resilience of L. monocytogenes in the environment might provide alternative views to tackle and apprehend new molecular mechanisms connected to bacterial pathogenesis. As a foodborne pathogen and in the context of food microbiology, the consideration that the ecological niche of this species oscillates between a mammalian host and the environment is particularly relevant but has hardly ever been taken into full consideration. As active molecules interfacing the cell and its surroundings, cell surface and extracellular proteins must play a key role in such interactions.

research articles

Comprehensive Exoproteome of L. monocytogenes EGD-e

Acknowledgment. This work was supported in part by the “Institut National de la Recherche Agronomique (INRA)”, the “Ministe`re de l’Agriculture et de la Peˆche” (DGAL no A03/02), and the European Framework Programme 6 (FP6) with the ProSafeBeef (Advancing Beef Safety and Quality through Research and Innovation) research consortium. Dr. Emilie Dumas was a PhD Research Fellow granted by the “Ministe`re de l’Education Nationale, de l’Enseignement Supe´rieur et de la Recherche (MENESR)”. We are very grateful to Re´gine Talon for critical reading of the manuscript, her pertinent comments and helpful discussions. We thank Franck Giacomoni (INRA metabolism exploration platform) for providing a user-friendly interface for analysis of proteomic results and converting generated database into Mascot format. Excellent technical assistance of Brigitte Duclos is also acknowledged. We are thankful to the INRA MIGALE bioinformatics platform (http:// migale.jouy.inra.fr) for providing computational resources. Supporting Information Available: Complete results from bioinformatic analyses based on the rational strategy allowing prediction of exoproteins in L. monocytogenes EGD-e are supplied in Table 1S. Results of bioinformatic analysis on proteins found by proteomics in the extracellular fraction are given in Table 2S. Comparison of prediction results in L. monocytogenes EGD-e from previous genomic analyses or available databases dedicated to the identification of exoproteins is made available in Table 3S. Proteomic identification of proteins predicted as bearing a signal peptide is provided in Table 4S and compared when using our mature or a standard database. Theoretical 2-DE maps depicting in details exoproteins secreted via each of the protein secretion pathways here unravelled is provided as Figure 1S. Experimental 2-DE maps with detailed labeling of protein spots is supplied as Figure 2S. Database in Mascot format containing the predicted mature set of proteins expressed in L. monocytogenes EGD-e, which was here used in the course of proteomic analysis for proteinspot identification, is provided as DB-Mature-LmoEGDe v1.0. This material is available free of charge via the Internet at http://pubs.acs.org.

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