Comparative proteomics to evaluate multi drug resistance in Escherichia coli.

Drug resistance in food-borne bacterial pathogens is an almost inevitable consequence of the use of antimicrobial drugs, used either therapeutically or to avoid infections in food-producing animals. In the past decades, the spread and inappropriate use of antibiotics have caused a considerable increase of antibiotics to which bacteria have developed resistance and, moreover, bacteria are becoming resistant to more than one antibiotic simultaneously. Understanding mechanisms at the molecular level is extremely important to control multi-resistant strains and to develop new therapeutic strategies. In the present study, comparative proteomics was applied to characterize membrane and cytosolic proteome in order to investigate the regulation of protein expression in multi-resistance E. coli isolated from young never vaccinated water buffalo. Results highlighted differentially expressed proteins under multi drug resistance conditions giving new insights about mechanisms involved in resistance, as quorum sensing mechanisms, and suggesting possible novel bacterial targets to develop alternative antibiotic drugs.

Comparative proteomic analysis of serum from patients with systemic sclerosis and sclerodermatous GVHD. Evidence of defective function of factor H.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. METHODOLOGY: Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. PRINCIPAL FINDINGS: Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. CONCLUSIONS: The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage.

The relevance of carbon dioxide metabolism in Streptococcus thermophilus.

Streptococcus thermophilus is a major component of dairy starter cultures used for the manufacture of yoghurt and cheese. In this study, the CO(2) metabolism of S. thermophilus DSM 20617(T), grown in either a N(2) atmosphere or an enriched CO(2) atmosphere, was analysed using both genetic and proteomic approaches. Growth experiments performed in a chemically defined medium revealed that CO(2) depletion resulted in bacterial arginine, aspartate and uracil auxotrophy. Moreover, CO(2) depletion governed a significant change in cell morphology, and a high reduction in biomass production. A comparative proteomic analysis revealed that cells of S. thermophilus showed a different degree of energy status depending on the CO(2) availability. In agreement with proteomic data, cells grown under N(2) showed a significantly higher milk acidification rate compared with those grown in an enriched CO(2) atmosphere. Experiments carried out on S. thermophilus wild-type and its derivative mutant, which was inactivated in the phosphoenolpyruvate carboxylase and carbamoyl-phosphate synthase activities responsible for fixing CO(2) to organic molecules, suggested that the anaplerotic reactions governed by these enzymes have a central role in bacterial metabolism. Our results reveal the capnophilic nature of this micro-organism, underlining the essential role of CO(2) in S. thermophilus physiology, and suggesting potential applications in dairy fermentation processes.