Project description:A group of gram positive bacteria that share the characteristic of fermenting hexose sugars to lactic acid are generally referred to as lactic acid bacteria (LAB). Enterococcus faecalis is one of the widely studied LABs due to a multiutude of reasons. On the one hand, it plays an important role in dairy industry, being for example a starter in cheese cultures. On the other hand, it accounts for a large part of the infections caused by the LABs in hospital environments. During the past few years, it developed resistance against most of the major antibiotics. Here, in an attempt to study its adaptive metabolism, a glutamine synthetase mutant (∆glnA) of E. faecalis was subjected to pH shift and the results from the integrative analysis of its metabolic network were compared to those of the wild type. The proteome data generated in this study were used to constrain the genome-scale metabolic network at two pH level, aiming to reduce the solution space and improve the accuracy of model simulation. This data particularly helped to come up with a new design for the amino acid transport system in the genome-scale model, resulting in an accurate reproduction of the metabolic behaviour of E. faecalis.
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:Genome-scale models represent the link between an organism's genetic information and experimentally observable biological phenotypes. They facilitate metabolic engineering and the discovery of network properties such as the identification of novel drug targets. Most commonly, metabolite consumption data is used to limit the solution space, sometimes in combination with gene expression data. However, information about gene expression only poorly correlates with the abundance of the respective proteins within the cell. As such, we developed a method to map and integrate the whole-cell proteome into genome-scale models on the example of lactic acid bacteria (LAB). To the best of our knowledge, this work represents the first effort to integrate proteome data into genome-scale models on such a scale.
Project description:Genome-scale models represent the link between an organism's genetic information and experimentally observable biological phenotypes. They facilitate metabolic engineering and the discovery of network properties such as the identification of novel drug targets. Most commonly, metabolite consumption data is used to limit the solution space, sometimes in combination with gene expression data. However, information about gene expression only poorly correlates with the abundance of the respective proteins within the cell. As such, we developed a method to map and integrate the whole-cell proteome into genome-scale models on the example of lactic acid bacteria (LAB). To the best of our knowledge, this work represents the first effort to integrate proteome data into genome-scale models on such a scale .
Project description:MKN74 cells were infected for 24 h or 5 days. For the 24 h infection, 80% confluent MKN74 cells were washed three times in PBS and incubated in antibiotic free medium. Overnight-grown colonies of E. faecalis were collected and added to the MKN74 cell culture at a multiplicity of infection (MOI) of 50 bacteria per cell. Cultures were maintained at 37 C under a 5% CO2 humidified atmosphere. 5 day infections were carried out by treating 65% confluent MKN74 cells with E. faecalis at a MOI of 10. Every 24 h, cells were washed three times with PBS and fresh medium and bacteria were added. Control cells were processed similarly in the absence of bacteria.
Project description:This study was undertaken to identify how gene expression of the urothelial cells respond to Enterococcus infection over the course of infection. 239 hypervariable (HV) genes were found to vary significantly over the time points, indicating a biological role in infection. Correlational clustering showed these HV genes fell into distinct families indicating a defined sequence of events following infection. Early events (0-1.5 hours post infection) were associated with upregulation of not well-known but bladder-associated genes which represented early immune response, cytoskeleton remodeling and cell cycle. Up- and downregulated genes at the middle time period (1.5-8 hours post infection) represented a variety of processes, from immune response/suppression, cell cycle/apoptosis to metabolism and cytoskeleton remodeling. Several transcription factors point to multiple pathways activation. At the late time points (8-10 hours post infection) downregulated genes represented major events of cell death, matrix degradation and immune response decline. Confocal microscopy confirms major cell death at these time points. Several events and pathways, like immune response suppression or cytoskeleton remodeling via Wnt/β-catenin and/or Rho/Rac pathway, were identified throughout the time course of HUC infection by Enterococcus. Those may be new targets for preventing and/or cure Enterococcus caused pathology. Keywords: Enterococcus, time course, microarray, infection, gene expression
Project description:Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.
Project description:Second messenger nucleotides such as c-di-AMP are produced by bacteria in response to environmental stimuli and can contribute to the regulation of a number of cellular processes including osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. Here, as part of an effort to uncover the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis, we utilized high-throughout RNA Sequencing analysis to explore the impact of deletion of genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP).