RESUMEN
Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the OG1RF ΔsrtAΔhtrA background partially restores the observed defects of the ΔsrtAΔhtrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal resistance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.
Asunto(s)
Aminoaciltransferasas , Proteínas Bacterianas , Biopelículas , Cisteína Endopeptidasas , Enterococcus faecalis , Fimbrias Bacterianas , Chaperonas Moleculares , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Enterococcus faecalis/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Biopelículas/crecimiento & desarrollo , Membrana Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Virulencia/genética , Antibacterianos/farmacología , Ceftriaxona/farmacologíaRESUMEN
Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.