Tryptic Shaving of Staphylococcus aureus Unveils Immunodominant Epitopes on the Bacterial Cell Surface.

The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics and by fast evolution into new lineages that rapidly spread within the healthy human population. This calls for development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available for clinical implementation, the present studies were aimed at identifying new leads for their development. For this purpose, we profiled the cell-surface-exposed staphylococcal proteome under infection-mimicking conditions by combining two approaches for "bacterial shaving" with immobilized or soluble trypsin and subsequent mass spectrometry analysis of liberated peptides. In parallel, non-covalently cell-wall-bound proteins extracted with potassium thiocyanate and the exoproteome fraction were analyzed by gel-free proteomics. All data are available through ProteomeXchange accession PXD000156. To pinpoint immunodominant bacterial-surface-exposed epitopes, we screened selected cell-wall-attached proteins of S. aureus for binding of immunoglobulin G from patients who have been challenged by different types of S. aureus due to chronic wound colonization. The combined results of these analyses highlight particular cell-surface-exposed S. aureus proteins with highly immunogenic exposed epitopes as potential targets for development of protective anti-staphylococcal immunization strategies.

Growth condition-dependent cell surface proteome analysis of Enterococcus faecium.

The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid-log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was "shaved" using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI-bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi-resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 (http://proteomecentral.proteomexchange.org/dataset/PXD002497).

Genomics and Proteomics Provide New Insight into the Commensal and Pathogenic Lifestyles of Bovine- and Human-Associated Staphylococcus epidermidis Strains.

The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated fdr marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains.

The Staphylococcus aureus proteome.

Staphylococcus aureus is a Gram-positive commensal bacterium that is regarded as a major threat for modern health care systems. This relates both to the ability of S. aureus to overcome antibiotic therapy by developing high-level resistance against multiple antibiotics and this bacterium's extensive arsenal of virulence factors. Understanding the mechanisms of resistance and functional studies on stress and starvation responses are the main goals of proteomics in staphylococcal research. This review high-lights recent advances in gel-based and gel-free proteomics analyses of S. aureus and pinpoints the importance of location-specific proteomics studies targeting the cytosol, the membrane, the cell surface and the extracellular milieu in combination with integrated global proteome studies. Emerging hot topics in staphylococcal proteomics are discussed with special focus on in vivo proteomics, membrane vesicles, biofilm formation and the acquisition of absolute proteome data for systems biological modeling approaches.

Identification of some main Streptococcus iniae associated proteins: relationship.

The surface-associated proteins play a key role in bacterial physiology and pathogenesis, and are the major targets in the development of new vaccines. These proteins contribute to the adaptation of bacteria to different hosts and environments. To study differences at the genomic level, we first sequenced the whole genome of Streptococcus iniae from fish (IUSA-1 strain) and compared it to Streptococcus iniae from human (9117 strain), revealing a high similitude between both strains. To gain further insights into host- and environment-specific differences, we then studied proteins in silico and by High Performance Liquid Chromatography. This approach successfully identified 54 secreted and surface proteins, including several proteins involved in cell wall synthesis and transport of solutes, as well as proteins with yet unknown function. These proteins highlight as interesting targets for further investigation in the interaction between Streptococcus iniae and its environment. Results reported in this study have shown a first analysis about the predicted and experimental associated proteins of Streptococcus iniae isolated from two different hosts: human and fish.