[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
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.
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.
Full-text · Article · Oct 2014 · Journal of Proteomics
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Aug 2011 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: A novel and rational genomic strategy mimicked the protein exportation steps leading to the extracellular location of secreted proteins in monoderm bacteria. This truly secretomic analysis allowed defining a mature protein databank in L. monocytogenes EGD-e used for subsequent spot identification. Based on theoretical and empirical exoproteomes, this generic approach resulted in the first comprehensive appraisal of its exoproteins, providing information on listerial physiology in relation with its habitat and lifestyle.
Full-text · Article · Oct 2010 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · May 2009 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Aims: To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.
Methods and Results: Growth and survival curves were recorded in brain–heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 μg ml−1 phenol while a rapid decrease in cell viability occurred in the presence of 30 μg ml−1 phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 μg ml−1 phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability.
Conclusions: The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes. Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes.
Significance and Impact of Study: This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.
Full-text · Article · May 2008 · Journal of Applied Microbiology
[Show abstract][Hide abstract] ABSTRACT: The analysis of cell-envelope subproteomes of the saprophytic bacterium Staphylococcus xylosus was investigated by evaluating different methods. The final procedure developed to extract, solubilize, and separate by two-dimensional gel electrophoresis two fractions enriched in cell wall and membrane proteins, respectively, is described. A total of 168 protein spots, corresponding to 90 distinct proteins, was identified by mass spectrometry. Their predictive localization was analyzed using several bioinformatics tools.
Full-text · Article · Oct 2007 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2006 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: 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).