The HtrA chaperone monitors sortase-assembled pilus biogenesis inEnterococcus faecalis

Abstract

AbstractSortase-assembled pili contribute to virulence in many Gram-positive bacteria. InEnterococcus 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 ofE. faecalisis 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 ofcroRin the ΔsrtAΔhtrAbackground partially restores the observed defects of the ΔsrtAΔhtrAstrain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal tolerance ofE. faecalisagainst cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into theE. faecalisresponse to endogenous membrane perturbations.Author summaryTo explore the role of the HtrA chaperone/protease inE. faecalisoff-pathway pilus clearance, we deletedhtrAin anE. faecalisOG1RF ΔsrtAstrain known to retain polymerized pili on the cell membrane. Cells in the ΔsrtAΔhtrAbackground are hyper-piliated, possess altered morphology, and are more susceptible to cell envelope-targeting antibiotics as compared to the parent OG1RF strain. RNA sequencing of the ΔsrtAΔhtrAstrain revealed transcriptional changes reminiscent of a membrane stress response. This response was pilus-dependent and contained several members of the CroR regulon. Inactivation of the response regulator CroR in the ΔsrtAΔhtrAbackground restored (at least partially) piliation and cell morphology but not antibiotic susceptibility, linking CroR for the first time to pilus biogenesis and endogenous cell envelope stress.