Project description:Small non coding RNA molecules (sncRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sncRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. Enterococcus faecalis. We characterized 11 sncRNAs by tiling microarray analysis, 5’ and 3’ RACE-PCR, and Northern blot analysis. Six sncRNAs were specifically expressed at exponential phase, two sncRNAs were observed at stationary phase, and three were detected during both phases. This is the first experimental genome-wide identification of sncRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.
Project description:To further investigate the homeostatic response of E. faecalis to Fe exposure, we examine the whole-genome transcriptional response of wild-type (WT) exposed to non toxic Fe excess. This experiment correspond the work titled Transcriptomic response of Enterococcus faecalis to iron excess (work in preparation)
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).
Project description:To further investigate the homeostatic response of E. faecalis to Fe exposure, we examine the whole-genome transcriptional response of wild-type (WT) exposed to non toxic Fe excess. This experiment correspond the work titled Transcriptomic response of Enterococcus faecalis to iron excess (work in preparation) A four chip study using total RNA recovered from four separate wild-type cultures of Enterococcus faecalis OG1RF, two controls samples (N medium growth) and two iron samples (N medium gowth with 0.5 mM Fe-NTA). Each chip measures the expression level of 3,114 genome genes from Enterococcus faecalis strain V583 (A7980-00-01).
Project description:Small non coding RNA molecules (sncRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sncRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. Enterococcus faecalis. We characterized 11 sncRNAs by tiling microarray analysis, 5’ and 3’ RACE-PCR, and Northern blot analysis. Six sncRNAs were specifically expressed at exponential phase, two sncRNAs were observed at stationary phase, and three were detected during both phases. This is the first experimental genome-wide identification of sncRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen. Identification of sncRNA candidates transcribed by E. faecalis V583 was undertaken with two samples of cells harvested in mid-log growth phase and stationary phase after 24h of incubation at 37°C in M17 glucose media.
Project description:Enterococcus faecalis (E. faecalis) is a Gram-(+) opportunistic pathogen associated with predominantly nosocomial wound infections. E. faecalis has been shown to suppress or evade immune-mediated clearance by the immune system and promote persistent infection. Here, we sought to interrogate whether E. faecalis infection induces transcriptomic changes in the host at the single-cell resolution using a mouse excisional wound model. Keratinocyte, fibroblast, endothelial and immune cell populations reveal unique clusters in the infected wounds. Cell communication analysis discovered strong ligand-receptor interactions between macrophages and endothelial cells in the infected niche, whilst fibroblast and keratinocytes instruct healing cellular interactions in untreated skin wounds. We also identified differential terminal lineage driving genes, following RNA velocity in each condition. Together, results suggest that E. faecalis infection alters the skin transcriptome, which may help promote the chronicity of bacteria-infected wounds.
Project description:Analysis of changes in gene expression in Enterococcus faecalis OG1 delta-EF2638 mutant compared to wild-type OG1 strain. The deletion mutant has a growth defect when grown with aeration The mutant presented in this study is described and characterized in Vesic, D. and Kristich, C.J. 2012. A Rex-family transcriptional repressor influnces H2O2 accumulation by Enterococcus faecalis. (submitted for publication) Microarray analysis was done using RNA isolated from two independent cultures of wild-type Enterococcus faecalis OG1 and two independent cultres of Enterococcus faecalis OG1 delta-EF2638 mutant; each RNA sample was subjected to triplicate hybridization (technical replicates) . Microarrays were custom designed to investigate expression of ORFs in Enterococcus faecalis OG1RF genome. The arrays were designed based on the OG1RF annotation generated with the Rapid Annotation Using Subsystem Technology (RAST) server (Aziz et. al. 2008. BMC Genomics 9:75), as described in Frank et al (2012) Infect. Immun. 80:539. The aim was eighteen probe pairs per ORF, each of which is present in triplicate.
Project description:Enterococcus faecalis, a member of the human gastrointestinal microbiota, is a Gram-positive, opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can suppress or evade immune-mediated clearance by macrophages to promote persistent infection, although the exact mechanisms and bacterial factor(s) involved are not well-defined. In this study, we examined E. faecalis factor(s) involved in suppressing macrophage activation, as well the macrophage pathways modulated by E. faecalis to suppress activation. We observed that E. faecalis prevents ERK and p65 phosphorylation and reduces MyD88 expression leading to a reduction in NF-κB activity. We identified E. faecalis lactate dehydrogenase, which is important for lactic acid production by E. faecalis, to be necessary for macrophage suppression and demonstrated that E. faecalis lactate dehydrogenase-mediated immune suppression promotes E. coli survival during polymicrobial wound infection. Taken together, these results suggest that that E. faecalis-derived lactic acid is involved in macrophage subversion and may help to promote the virulence of co-infecting bacteria.