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[
Res Microbiol,
2017]
Phosphotransferase systems are common and essential in bacteria, which are in charge of sugar transportation and phosphorylation. However, phosphotransferase systems were found in recent years to be associated with environmental stress factors. This study investigated the role of the mannose/fructose/sorbose phosphotransferase systems in Enterococcus faecalis OG1RF in adaption to harsh environments by construction of pts mutants. More than one mannose/fructose/sorbose phosphotransferase system was found in E. faecalis OG1RF, and the elimination of pts gene at different loci generated different after-effects corresponding to different ambiences. An in vitro study showed that the presence of intact phosphotransferase systems in E. faecalis OG1RF promoted resistance to hydrogen peroxide and acid and enhanced susceptibility to pediocin. In vivo study demonstrated that the presence of intact phosphotransferase systems induced more hazardous substances like superoxide dismutase (SOD) in Caenorhabditis elegans and enhanced bacterial infection and survival in macrophages J774A.1 and BMM. In addition, phosphotransferase systems regulated transcription of antioxidant and catabolite genes such as katA, gor, lysR, hypR, rex, hprK and tpx to different extent s(-6.3-to 3.5-fold). It is therefore suggested that pts genes are regulatory factors promoting adaption of E. faecalis OG1RF to stressful conditions, thereby enhancing the possibility of bacterial survival and infectivity.
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Benedetto, Alexandre, Hardgrave, Alex, Urbaniak, Mick, Whittingham-Dowd, Jayde, Zarate Potes, Alejandra, Cabreiro, Filipe, Parry, Jackie, Gems, David, Fathallah, Nadin, Kosztelnik, Monika, Martin, Jack, Cetnar, Kalina, Norvaisas, Povilas, Au, Catherine, Rezwana, Ruhi
[
International Worm Meeting,
2021]
Key words Kynurenine pathway, E. faecalis, gut infection, microbiota, lysosome-related organelles autofluorescence. Abstract The kynurenine pathway (KP), main catabolic route for the essential amino-acid tryptophan, is well-known for its immunomodulatory role in mammals. While investigating death fluorescence in C. elegans, anthranilic acid (AA)-loaded lysosome-related organelles (LROs) were previously found responsible for the blue auto-fluorescence seen in the worm gut (Coburn et al. PLOS Biol. 2013). Given the bacteriostatic potential of AA and other kynurenine pathway compounds, we hypothesised that LROs and the KP play a key role in C. elegans gut microbial control. To test this idea, we exposed C. elegans to a worm-pathogenic strain of E. faecalis (OG1RF) and observed changes in gut morphology and autofluorescence dynamics upon infection. Transcriptomics and targeted metabolomics analyses further showed that KP activity is modulated upon E. faecalis infection. Using a combination of KP mutants from the Nollen lab (Van Der Goot et al. PNAS 2012), we observed that inhibition of various KP enzymes differentially affect C. elegans resistance to E. faecalis infection. E. faecalis growth on KP mutant worm extracts confirmed that resistant mutants produce bacteriostatic compounds, which we measured by HPLC. This was verified by the delayed or reduced gut colonisation of OG1RF-GFP (gifted by D. Garsin), and the ability for some mutants to thrive on OG1RF loans. We are currently investigating a broader role for the KP in C. elegans gut microbiota control, notably using newly generated CeMBio fluorescent strains.
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[
J Microbiol Methods,
2012]
Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.
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Haller D, Lengfelder I, Steck N, Sartor RB, Bao Y, Hansen JJ, Murray BE, Tchaptchet S, Ocvirk S, Huebner J, Lagkouvardos I, Roh JH, Carroll IM, Sava IG
[
PLoS Pathog,
2015]
The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2+/-1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (epaB score: 4.7+/-2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (lgt score: 2.3+/-2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae and impaired penetration into the colonic mucus layer of IL-10-/- mice. Lipoprotein-deficient E. faecalis exhibited an impaired TLR2-mediated activation of BMDCs in vitro despite their ability to fully reactivate MLN cells as well as MLN-derived colitogenic T cells ex vivo. E. faecalis virulence factors accounting for bacterial adhesion to mucosal surfaces as well as intestinal barrier disruption partially contribute to colitogenic activity of E. faecalis. Beyond their well-known role in infections, cell surface-associated lipoproteins are essential structures for colitogenic activity of E. faecalis by mediating innate immune cell activation.