Ingrid Chafsey

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.

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.

Response of Listeria monocytogenes to liquid smoke

Aims:  To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.

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.

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

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. 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.

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.

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

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).

How different is the proteome of the extended spectrum β-lactamase producing Escherichia coli strains from seagulls of the Berlengas natural reserve of Portugal?

UNLABELLED β-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Escherichia coli strains 5A, 10A, 12A and 23B isolated from Seagulls feces, are cefotaxime-resistant strains that produces extended-spectrum beta-lactamases. Bacterial resistance to these antibiotics occurs predominantly through structural modification on the penicillin-binding proteins and enzymatic inactivation by extended-spectrum β-lactamases. Using classical proteomic techniques (2D-GE) coupled to mass spectrometry and bioinformatics extended analysis, in this study, we report several significant differences in cytoplasmic proteins expression when the strains were submitted to antibiotic stress and when the resistant strains were compared with a non-resistant strain. A total of 79 differentially expressed spots were collected for protein identification. Significant level of expression was found in antibiotic resistant proteins like β-lactamase CTX-M-1 and TEM and also in proteins related with oxidative stress. This approach might help us understand which pathways form barriers for antibiotics, another possible new pathways involved in antibiotic resistance to devise appropriate strategies for their control already recognized by the World Health Organization and the European Commission. BIOLOGICAL SIGNIFICANCE This study highlights the protein differences when a resistant strain is under antibiotic pressure and how different can be a sensible and resistant strain at the protein level. This survey might help us to understand the specifics barriers for antibiotics and which pathways are involved in its resistance crosswise the wildlife.

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

UNLABELLED 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.

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

ABSTRACT 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.