Specific Proteomic Identification of Collagen-Binding Proteins in Escherichia coli O157:H7: Characterisation of OmpA as a Potent Vaccine Antigen.

Escherichia coli is a versatile commensal species of the animal gut that can also be a pathogen able to cause intestinal and extraintestinal infections. The plasticity of its genome has led to the evolution of pathogenic strains, which represent a threat to global health. Additionally, E. coli strains are major drivers of antibiotic resistance, highlighting the urgent need for new treatment and prevention measures. The antigenic and structural heterogeneity of enterohaemorrhagic E. coli colonisation factors has limited their use for the development of effective and cross-protective vaccines. However, the emergence of new strains that express virulence factors deriving from different E. coli diarrhoeagenic pathotypes suggests that a vaccine targeting conserved proteins could be a more effective approach. In this study, we conducted proteomics analysis and functional protein characterisation to identify a group of proteins potentially involved in the adhesion of E. coli O157:H7 to the extracellular matrix and intestinal epithelial cells. Among them, OmpA has been identified as a highly conserved and immunogenic antigen, playing a significant role in the adhesion phenotype of E. coli O157:H7 and in bacterial aggregation. Furthermore, antibodies raised against recombinant OmpA effectively reduced the adhesion of E. coli O157:H7 to intestinal epithelial cells. The present work highlights the role of OmpA as a potent antigen for the development of a vaccine against intestinal pathogenic E. coli.

Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

The Secretome landscape of Escherichia coli O157:H7: Deciphering the cell-surface, outer membrane vesicle and extracellular subproteomes.

Among diarrheagenic E. coli (DEC), enterohaemorrhagic E. coli (EHEC) are the most virulent anthropozoonotic agents. The ability of bacterial cells to functionally interact with their surrounding essentially relies on the secretion of different protein effectors. To experimentally determine the repertoire of extracytoproteins in E. coli O157:H7, a subproteomic analysis was performed not only considering the extracellular milieu but the cell surface and outer membrane vesicles. Following a secretome-based approach, the proteins trafficking from the interior to the exterior of the cell were depicted considering cognate protein transport systems and subcellular localisation. Label-free quantitative analysis of the proteosurfaceome, proteovesiculome and exoproteome from E. coli O157:H7 grown in three different nutrient media revealed differential protein expression profiles and allowed defining the core and variant subproteomes. Network analysis further revealed the higher abundance of some protein clusters in chemically defined medium over rich complex medium, especially related to some outer membrane proteins, ABC transport and Type III secretion systems. This first comprehensive study of the EHEC secretome unravels the profound influence of environmental conditions on the extracytoplasmic proteome, provides new insight in the physiology of E. coli O157:H7 and identifies potentially important molecular targets for the development of preventive strategies against EHEC/STEC. SIGNIFICANCE: Escherichia coli O157:H7 is responsible for severe diarrhoea especially in young children. Despite years of investigations, the global view of the extracytoplasmic proteins expressed in this microorganism was eluded. To provide the first comprehensive view of the secretome landscape of E. coli O157:H7, the exoproteome, proteosurfaceome and proteovesiculome were profiled using growth conditions most likely to induce changes in bacterial protein secretion. The profound influence of growth conditions on the extracytoplasmic proteome was unravelled and allowed identifying the core and variant subproteomes. Besides new insight in the physiology of enterohaemorrhagic E. coli, these proteins potentially constitute important molecular targets for the development of preventive strategies.

Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Differential biotin labelling of the cell envelope proteins in lipopolysaccharidic diderm bacteria: Exploring the proteosurfaceome of Escherichia coli using sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin.

Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species. While generally pass over in silence, some periplasmic proteins, but also some inner membrane lipoproteins, integral membrane proteins and cytoplasmic proteins (cytoproteins) are systematically identified following this approach. To limit cell lysis and diffusion of the sulfo-NHS-SS-biotin through the outer membrane, biotin labelling was tested over short incubation times and proved to be as efficient for 1 min at room temperature. To further limit labelling of protein located below the outer membrane, the use of high-molecular weight sulfo-NHS-PEG4-bismannose-SS-biotin appeared to recover differentially cell-envelope proteins compared to low-molecular weight sulfo-NHS-SS-biotin. Actually, the sulfo-NHS-SS-biotin recovers at a higher extent the proteins completely or partly exposed in the periplasm than sulfo-NHS-PEG4-bismannose-SS-biotin, namely periplasmic and integral membrane proteins as well as inner membrane and outer membrane lipoproteins. These results highlight that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria. SIGNIFICANCE: While generally pass over in silence, some periplasmic proteins, inner membrane lipoproteins (IMLs), integral membrane proteins (IMPs) and cytoplasmic proteins (cytoproteins) are systematically identified following cell-surface biotin labelling in lipopolysaccharidic diderm bacteria (archetypal Gram-negative bacteria). The use of biotinylation molecules of different sizes, namely sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin, was demonstrated to provide a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria.

Subproteomic signature comparison of in vitro selected fluoroquinolone resistance and ciprofloxacin stress in Salmonella Typhimurium DT104B.

BACKGROUND: Fluoroquinolone resistance in nontyphoidal Salmonella is a situation of serious and international concern, particularly in S. Typhimurium DT104B multiresistant strains. Although known to be multifactorial, fluoroquinolone resistance is still far from a complete understanding. METHODS: Subproteome changes between an experimentally selected fluoroquinolone-resistant strain (Se6-M) and its parent strain (Se6), and also in Se6-M under ciprofloxacin (CIP) stress, were evaluated in order to give new insights into the mechanisms involved. Proteomes were compared at the intracellular and membrane levels by a 2-DE~LC-MS/MS and a shotgun LC-MS/MS approach, respectively. RESULTS: In total, 35 differentially abundant proteins were identified when comparing Se6 with Se6-M (25 more abundant in Se6 and 10 more abundant in Se6-M) and 82 were identified between Se6-M and Se6-M+CIP (51 more abundant in Se6-M and 31 more abundant under ciprofloxacin stress). CONCLUSION: Several proteins with known and possible roles in quinolone resistance were identified which provide important information about mechanism-related differential protein expression, supporting the current knowledge and also leading to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.

Comparative subproteomic analysis of clinically acquired fluoroquinolone resistance and ciprofloxacin stress in Salmonella Typhimurium DT104B.

PURPOSE: Antimicrobial resistance is a worldwide public health threat and Salmonella enterica subsp. enterica serotype Typhimurium phage type DT104B multiresistant strains with additional quinolone resistance have been responsible for global outbreaks and high mortality. Quinolone resistance is known to be multifactorial but is still far from a complete understanding. To give new insights about the resistance mechanisms involved, this work aimed to evaluate subproteome changes between an S. Typhimurium DT104B clinical strain that acquired fluoroquinolone resistance after treatment (Se20) and its pretreatment parental strain (Se6), and also subproteome variations in Se20 under ciprofloxacin (CIP) stress (Se20+CIP). EXPERIMENTAL DESIGN: The proteomes were compared at the intracellular and membrane levels by a 2-DE∼LC-MS/MS and a shotgun LC-MS/MS approach, respectively. RESULTS: In total, 14 differentially abundant proteins were identified when comparing Se6 with Se20 and 91 were identified between Se20 and Se20+CIP. Several proteins with known and possible roles in quinolone resistance (AAC(6')-Ib-cr4, OmpD, OmpX, GlmS, GlmU, H-NS, etc.) were identified and discussed. CONCLUSIONS AND CLINICAL RELEVANCE: The great number of proteins identified in this study provides important information about mechanism-related differential protein expression which supports the current knowledge and might lead to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.

Impacts of experimentally induced and clinically acquired quinolone resistance on the membrane and intracellular subproteomes of Salmonella Typhimurium DT104B.

UNLABELLED: Antimicrobial resistance is a growing public health threat worldwide that is still far from a complete understanding. Salmonella Typhimurium DT104 multiresistant strains with additional quinolone resistance are highly adaptive and have been responsible for global outbreaks and high mortality. In order to give new insights about the resistance mechanisms involved, the developed work aimed to point out subproteome changes between a DT104B clinical strain (Se20) that acquired quinolone resistance after patient treatment and an in vitro induced clonally related highly-resistant mutant (Se6-M). The intracellular subproteomes were compared by a 2-DE/LC-MS/MS approach and a total of 50 unique proteins were identified (32 more abundant in Se20 and 18 more abundant in Se6-M). The membrane subproteomes were analysed by a shotgun LC-MS/MS approach, where 7 differentially abundant proteins were identified (5 more abundant in Se6-M and 2 more abundant in Se20). Several proteins known to be directly related to quinolone resistance mechanisms (AAC(6')-Ib-cr4, OmpC, OmpD, OmpX, etc.) and MipA, recently reported as novel antibiotic resistance-related protein, were identified. Other proteins (Fur, SodA, SucB, AtpD/AtpG, OmpC, GltI, CheM/CheB, etc.) reflecting the metabolic re-adjustments occurred in each strain in order to acquire quinolone resistance were also identified. Moreover, proteins involved in lipopolysaccharide biosynthesis (RfbF, RfbG, GmhA) and export (LptA) were detected, supporting the importance of exploring these proteins as targets for the development of new antimicrobial agents. In conclusion, this study provides new insights into the mechanisms involved in the acquisition of antibiotic resistance, which can be highly valuable for the development of improved therapeutic strategies. BIOLOGICAL SIGNIFICANCE: This comparative proteomic study revealed a large number of differentially regulated proteins involved in antibiotic resistance which can be of great value to drug discovery, research and development programmes.

Contribution of the multiple Type I signal peptidases to the secretome of Listeria monocytogenes: deciphering their specificity for secreted exoproteins by exoproteomic analysis.

As commonly seen in monoderm bacteria, Listeria monocytogenes possesses multiple membrane-bound signal peptidases of Type I (SPases I) called SipX, SipY and SipZ. In order to decipher their respective contribution in an integrated and global view, the complement of the secretome corresponding to the exoproteome was resolved by two-dimensional gel electrophoresis (2-DE). This was performed for L. monocytogenes sipX(-), sipY(-), sipZ(-) single mutants, as well as for ΔsipXY and ΔsipYZ double mutants, and then compared to that of the wild type strain. Remarkably, the amounts of listeriolysin O (LLO), phosphatidylcholine phospholipase C (PlcB) and zinc metalloproteinase Mpl in the extracellular milieu were significantly decreased upon inactivation of SipZ. For the majority of the Sec-secreted exoproteins identified, protein secretion was not affected by the inactivation of one or two of the SPases I, supporting the concept that the three SPases I have overlapping specificities for the cleavage of the signal peptides. The current study reveals that the role of SipZ as the major SPase I of L. monocytogenes applies only to a small subset of the secreted exoproteins. Rather than absolute, the notion of major and minor SPases thus appears to be relative. In addition to new insight into bacterial physiology, this investigation of the contribution of the SPases I to the exoproteome of L. monocytogenes paves the way for further characterization of other complements of the secretome under various environmental conditions. BIOLOGICAL SIGNIFICANCE: L. monocytogenes encodes three orthologous signal peptidases of Type I (SPases I). SipZ improves the secretion efficiency for a subset of extracellular virulence factors. Multiple SPases I are functionally redundant for the majority of the Sec-secreted exoproteins of L. monocytogenes. The concepts of major and minor SPases are not absolute but relative.

Surfaceome and exoproteome of a clinical sequence type 398 methicillin resistant Staphylococcus aureus strain.

For many years Staphylococcus aureus has been recognized as an important human pathogen. In this study, the surfacome and exoproteome of a clinical sample of MRSA was analyzed. The C2355 strain, previously typed as ST398 and spa-t011 and showing a phenotype of multiresistance to antibiotics, has several resistance genes. Using shotgun proteomics and bioinformatics tools, 236 proteins were identified in the surfaceome and 99 proteins in the exoproteome. Although many of these proteins are related to basic cell functions, some are related to virulence and pathogenicity like catalase and isdA, main actors in S. aureus infection, and others are related to antibiotic action or eventually resistance like penicillin binding protein, a cell-wall protein. Studying the proteomes of different subcellular compartments should improve our understanding of this pathogen, a microorganism with several mechanisms of resistance and pathogenicity, and provide valuable data for bioinformatics databases.

Effect of vancomycin on the proteome of the multiresistant Enterococcus faecium SU18 strain.

Enterococci are not highly pathogenic bacteria, but the incidence of vancomycin resistance among clinical isolates of this microbial group is steadily increasing, posing a threat to public health. Vancomycin-resistant enterococci are currently some of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. To understand the molecular mechanisms of bacterial resistance to glycopeptides, we obtained proteomic profiles of the vancomycin-resistant Enterococcus faecium SU18 strain treated with and without vancomycin. Fourteen proteins were differentially expressed in SU18, seven of which were up-regulated and seven down-regulated. Proteins involved in the vancomycin resistance mechanism, such as the VanA protein, VanA ligase, VanR and D-Ala-D-Ala dipeptidase, were up-regulated in the presence of vancomycin, while metabolism-related proteins, such as triosephosphate isomerase, guanine monophosphate synthase and glyceraldehyde-3-phosphate dehydrogenase were down-regulated. Overall the compensatory response of SU18 to antibiotics is to alter expression of proteins related to antibiotic resistance, cell wall formation and energy metabolism. Some of the differentially expressed proteins might enhance antimicrobial activity and are now being investigated as potential therapeutic drug targets in other pathogenic bacteria. BIOLOGICAL SIGNIFICANCE: This study highlights the power of proteomics in the study of differential protein expression in a multiresistant Enterococcus faecium strain when subjected to vancomycin stress.

Comprehensive appraisal of the extracellular proteins from a monoderm bacterium: theoretical and empirical exoproteomes of Listeria monocytogenes EGD-e by secretomics.

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.

Comparative subproteome analyses of planktonic and sessile Staphylococcus xylosus C2a: new insight in cell physiology of a coagulase-negative Staphylococcus in biofilm.

Staphylococcus xylosus is a Gram-positive bacterium found on the skin of mammals and frequently isolated from food plants and fermented cheese or meat. To gain further insight in protein determinants involved in biofilm formation by this coagulase-negative Staphylococcus, a comparative proteomic analysis between planktonic and sessile cells was performed. With the use of a protocol previously developed, protein patterns of the cytoplasmic and cell envelope fractions were compared by 2-DE. Following protein identification by MALDI-TOF mass spectrometry and bioinformatic analyses, this study revealed differences in expression levels of 89 distinct proteins with 55 up-expressed and 34 down-expressed proteins in biofilm compared to planktonic cells. Most proteins differentially expressed were related to nitrogen and carbon metabolisms. Besides amino acid biosynthesis and protein translation, protein determinants related to protein secretion were up-expressed in biofilm, suggesting a more active protein trafficking in sessile cells. While up-expression of several enzymes involved in pentose phosphate and glycolytic pathways was observed in biofilm, connections with unexpected metabolic routes were further unravelled. Indeed, this proteomic analysis allowed identifying novel proteins that could be involved in a previously uncovered exopolysaccharide biosynthetic pathway in S. xylosus as well as several enzymes related to polyketide biosynthesis. This findings are particularly relevant considering exopolysaccharide production in S. xylosus is ica-independent contrary to coagulase-negative model strain Staphylococcus epidermidis RP62A.

Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative staphylococcus.

Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a.

Adapted tolerance to benzalkonium chloride in Escherichia coli K-12 studied by transcriptome and proteome analyses.

Benzalkonium chloride (BC) is a commonly used disinfectant and preservative. This study describes changes in expression level at the transcriptomic and proteomic level for Escherichia coli K-12 gradually adapted to a tolerance level to BC of 7-8 times the initial MIC. Results from DNA arrays and two-dimensional gel electrophoresis for global gene and protein expression studies were confirmed by real-time quantitative PCR. Peptide mass fingerprinting by MALDI-TOF MS was used to identify differentially expressed proteins. Changes in expression level in adapted cells were shown for porins, drug transporters, glycolytic enzymes, ribosomal subunits and several genes and proteins involved in protection against oxidative stress and antibiotics. Adapted strains showed increased tolerance to several antibiotics. In conclusion, E. coli K-12 adapted to higher tolerance to BC acquired several general resistance mechanisms, including responses normally related to the multiple antibiotic resistance (Mar) regulon and protection against oxidative stress. The results revealed that BC treatment might result in superoxide stress in E. coli.

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure.

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.

Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence.

In this study, the role of Listeria monocytogenes ferritin was investigated. The fri gene encoding the ferritin was deleted and the phenotype of the mutant was analyzed demonstrating that ferritin is necessary for optimal growth in minimal medium in both presence and absence of iron, as well as after cold- and heat-shock. We also showed that ferritin provides protection against reactive oxygen species and is essential for full virulence of L. monocytogenes. A comparative proteomic analysis revealed an effect of the fri deletion on the levels of listeriolysin O and several stress proteins. Together, our study demonstrates that fri has multiple roles that contribute to Listeria virulence.

Two-dimensional electrophoresis database of Listeria monocytogenes EGDe proteome and proteomic analysis of mid-log and stationary growth phase cells.

Listeria monocytogenes is the causative agent of listeriosis, one of the most significant foodborne diseases in industrialized countries. The complete genome of the L. monocytogenes EGDe strain, belonging to the serogroup 1/2a, has been sequenced and is comprised of 2853 open reading frames. The objective of the current study was to construct a two-dimensional (2-D) database of the proteome of this strain. The soluble protein fractions of the microorganism were recovered either in the mid-log or in the stationary phase of growth at 37 degrees C. These fractions were analyzed by 2-D electrophoresis (2-DE), using immobilized pH gradient strips of various pH values (3-10, 3-6, and 5-8) for the first-dimensional separations and 12.5% acrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 201 protein spots corresponding to 126 different proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The 2-DE maps presented here provide a first basis for further investigations of protein expression in L. monocytogenes. In this way, the comparison of proteome between cells in the exponential or stationary phase of growth at 37 degrees C allowed us to characterize 161 variations in protein spot intensity, of which 38 were identified. Among the differentially expressed proteins were ribosomal proteins (RpsF, RplJ, and RpmE), proteins involved in cellular metabolism (GlpD, PdhD, Pgm, Lmo1372, Lmo2696, and Lmo2743) or in stress adaptation (GroES and ferritin), a fructose-specific phosphotransferase enzyme IIB (Lmo0399) and different post-translational modified forms of listeriolysin (LLO).