Project description:The enterococci comprise a genus of 49 low-GC content Gram-positive commensal species within the Firmicutes phylum that are known to occupy diverse habitats, notably the gastrointestinal core microbiota of nearly every phylum, including human. Of particular clinical relevance are two rogue species of enterococci, Enterococcus faecalis and the distantly related Enterococcus faecium, standing among the nefarious multi-drug resistant and hospital-acquired pathogens. Despite increasing evidence for RNA-based regulation in the enterococci, including regulation of virulence factors, their transcriptome structure and arsenal of regulatory small sRNAs (sRNAs) are not thoroughly understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptomes of E. faecalis V583 and E. faecium AUS0004. We identified 2517 and 2771 transcription start sites (TSS) in E. faecalis and E. faecium, respectively. Based on the identified TSS, we created a global map of s70 promoter motifs. We also revealed features of 5’ and 3’UTRs across the genomes. The transcriptome maps also predicted 150 and 128 sRNA candidates in E. faecalis and E. faecium, respectively, some of which have been identified in previous studies and many of which are new. Finally, we validated several of the predicted sRNAs by Northern Blot in biologically relevant conditions. Comprehensive TSS mapping of two representative strains will provide a valuable resource for the continued development of RNA biology in the Enterococci.
Project description:Enterococci are normal inhabitants of the gastrointestinal tracts of humans and animals and thanks to their capability to tolerate different environmental conditions and their high rates of gene transfer, they are able to colonize various ecological niches, as food matrices. Enterococcus faecalis bacteria are defined as “border line” microorganisms. From one side they are used as food starters, bio-control agents and probiotics to improve human or animal health. From the other side, in the last 2 two decades enterococci have emerged as important nosocomial pathogens, because bearing high-level of resistance to antibiotics and several putative virulence factors. In this study the proteomic (LC-MS/MS) and the phenotypic characterization (enzymatic methods) of three strains of E. faecalis with different origin were performed in order to investigate the differences and/or similarities occurring between pathogenic and health promoting bacteria. The E. faecalis D27 isolated as cheese contaminant, E. faecalis Symbioflor 1 a probiotic strain and E. faecalis UW3114 a clinical isolate involved in urinary tract infection were the objects of the study. The comparison of cytosolic protein expression profiles of the three strains, highlighted statistically significant changes in the abundance of proteins mainly involved in specific metabolic pathways, nutrient transport, stress response and cell wall modulation. Moreover, especially in the food contaminant and the clinical isolate, several proteins with potential pathogenic implications were found. The analysis of the extracellular proteome provided interesting results concerning proteins involved in bacterial communication, such as pheromones and conjugative elements and also proteins able to interact with human components. The phenotypic assays evaluating i) biofilm formation ii) hemolytic activity on blood agar plates iii) protease activity, allowed typing of bacteria in relation to well-known pathogenic traits. The obtained results confirmed the pathogenic profile associated to the clinical isolate as compared to the food contaminant and to the probiotic and allowed to elucidate the risks associated with the poor characterized foodborne E. faecalis D27.
Project description:The biofilm proteome profile of endodontic and systemic pathogen E. faecalis has been analysed in this study to identify markers associated with its biofilm formation. Strong and weak biofilm forming E. faecalis clinical isolates were compared with two standard ATCC strains of E. faecalis in order to elucidate the biological pathways associated with the biofilm formation capability of E. faecalis.