show Abstracthide AbstractSortase-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 ?srtA?htrA background partially restores the observed defects of the ?srtA?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. Overall design: To investigate the relationship between HtrA, pilus biogenesis, and the CroRS two-component system in E. faecalis, we created a panel of clean deletion mutant strains of htrA, srtA, htrA srtA, croR::tn htrA srtA, and srtA htrA ebpABC, and performed RNA-Seq on exponentially grown cells in TSB supplemented with 0.25% glucose. We also performed RNA-Seq of wild-type OG1RF treated with the cell envelope-targeting antibiotic daptomycin and compared it to untreated. In this case, cells were grown in BHI media.