The HtrA chaperone monitors sortase-assembled pilus biogenesis in Enterococcus faecalis

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 Δ srtA Δ htrA background partially restores the observed defects of the Δ srtA Δ htrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal tolerance 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. Author summary To explore the role of the HtrA chaperone/protease in E. faecalis off-pathway pilus clearance, we deleted htrA in an E. faecalis OG1RF Δ srtA strain known to retain polymerized pili on the cell membrane. Cells in the Δ srtA Δ htrA background 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 Δ srtA Δ htrA strain 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 Δ srtA Δ htrA background 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. ### Competing Interest Statement The authors have declared no competing interest.