Colomer Winter, C. | bioRxiv Science

bioRxiv2025

Fsr quorum sensing system restricts biofilm growth and activates inflammation in enterococcal infective endocarditis

Infective endocarditis (IE) is a life-threatening biofilm-associated infection, yet the factors driving biofilm formation remain poorly understood. Here, we identified the Fsr quorum sensing (QS) system of Enterococcus faecalis as a potent negative regulator of IE pathogenesis. Using microfluidic and in vivo models, we show that Fsr is induced in late IE when bacteria become shielded from blood flow. Deleting Fsr altered biofilm metabolism and promoted robust biofilm growth and gentamicin tolerance in vivo. Furthermore, Fsr inactivation attenuated inflammation by disrupting IL-1{beta} cleavage and activation via the Fsr-regulated gelatinase (gelE), allowing biofilm to grow unchecked by the immune system. Consistent with our pre-clinical findings, analysis of two IE patient cohorts linked naturally occurring Fsr-deficient E. faecalis to prolonged bacteremia. Overall, our findings provide insights into the role of QS in biofilm growth, persistence, and immune evasion in enterococcal IE.

microbiology↗

bioRxiv2023

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 {Delta}srtA{Delta}htrA background partially restores the observed defects of the {Delta}srtA{Delta}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 summaryTo explore the role of the HtrA chaperone/protease in E. faecalis off-pathway pilus clearance, we deleted htrA in an E. faecalis OG1RF {Delta}srtA strain known to retain polymerized pili on the cell membrane. Cells in the {Delta}srtA{Delta}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 {Delta}srtA{Delta}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 {Delta}srtA{Delta}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.

microbiology↗