HtrA and CroRS two-component signal transduction system monitor sortase-assembled pilus biogenesis in Enterococcus faecalis

Enterococcus faecalis is one of the leading causes of hospital-acquired infections (HAIs). One of the most commonly associated virulence factors in these HAIs is the endocarditis and biofilm-associated pilus (Ebp) which is often implicated in biofilm formation. E. faecalis is associated with urinary tract infections (UTI), bacteremia, endocarditis, surgical wounds, and many other infections. These infections likely give rise to unfolded or misfolded proteins, leading to the accumulation of potentially harmful protein aggregates. E. faecalis must evolve strategies to combat these stresses encountered during colonization and infection. It is not known how these misfolded proteins, including Ebp, are processed. In this study, we determined the contribution of a highly conserved serine protease, HtrA, to membrane stress tolerance in E. faecalis. We found that HtrAEF is not involved in growth or survival under a wide range of stress conditions, nor is it involved in biofilm formation. However, the ∆htrA mutant was attenuated in a competitive murine wound infection, suggesting its contribution to colonization and infection in host tissues. In many bacteria, HtrA deals with accumulated protein stresses and/or is part of a proteolytic pathway. The absence of HtrA alone does not result in accumulation of proteins. We postulated that the induction of protein accumulation in the absence of HtrA in E. faecalis may reveal a true role for HtrAEF. We created a ∆srtA∆htrA to mimic membrane stress where sortase substrates, such as Ebp pili, accumulate on the cell membrane. In this mutant background, we observed hyperpiliation as compared to single mutants. Furthermore, ∆srtA∆htrA formed chains and displayed aberrant placement of septa in addition to increased pilus expression. ∆srtA∆htrA cells appeared coccoid and wider at the cell width than WT. In addition, this phenotype was pilus-dependent, as we no longer observed these aberrant cell morphologies in a ∆srtA∆ebpABC∆htrA strain. Transcriptomic analysis revealed upregulation of the CroR-CroS two-component system (TCS) in altering cell shape in a ∆srtA∆htrA mutant strain. Deletion of croR in ∆srtA∆htrA resulted in a reversal of cell morphology and piliation level to WT, suggesting that CroR mediates reparative transcriptional responses during membrane stress. Global expression analysis using RNA-seq identified possible CroR target genes, suggesting a possible novel role of this TCS in E. faecalis pilus biogenesis. This thesis is the first comprehensive study of HtrA in E. faecalis. We have discovered novel roles for HtrA and the CroR-CroS TCS in monitoring pilus biogenesis, providing potential inhibitory targets to limit the biogenesis of this important virulence factor.Doctor of Philosoph