Enterococcus faecalis and Candida albicans are considered by the CDC to be among the most serious global antimicrobial resistant threats. While both species normally exist as human commensals, both are the causative agents of several oral diseases due to high-levels of resistance to common forms of treatment. Currently, most antimicrobials target essential processes in generating cell wall and/or membrane, however generation of resistance remains a major threat to a successful outcome of treatment. Therefore, therapies that target a virulence trait rather than an essential process may be a more desirable approach towards combating the development of resistance. Preliminary results indicate that the E. faecalis thiodredoxin DsbA has a previously unrealized role in catalyzing oxidative protein folding, and is required for virulence. The objective of this research proposal is to determine the contribution of DsbA towards E. faecalis pathogenicity. The project will test the hypothesis that DsbA is required for the post-translational disulfide bond formation of EntV, a bacteriocin implicated in the suppression of C. albicans hyphal morphogenesis, and in additional proteins required for virulence.
Aim 1 will determine the contribution of DsbA towards EntV maturation. Mutations in disulfide bonding cysteines will be inserted into EntV to determine the effect on its function. In addition, we will test if DsbA is the source of disulfide bond formation in EntV. Additional proteins that are negatively affected by the loss of dsbA, and that are required for virulence will be tested in Aim 2. First, transposon (Tn) insertion mutants of proteins predicted to require DsbA will be visualized by transmission electron microscopy and morphology compared to the dsbA deletion mutant. In addition, biofilm formation on various media will also be evaluated. To determine the contribution of the newly identified protein(s) toward E. faecalis virulence, Tn- mutants will be tested within a C. elegans model of infection. Next, in frame deletion mutants of proteins predicted to require DsbA for post-translational disulfide bond formation will be generated, and tested in a similar fashion as the Tn-mutants to confirm our original observations. Mutations in disulfide bonding cysteines will then be inserted into identified proteins to determine the effect on their function. Next, DsbA will be tested to determine if it is the source of disulfide bond formation in identified proteins. Finally, to confirm that the newly identified DsbA substrates contribute to E. faecalis virulence, each deletion mutant will be tested within a mouse model of peritonitis. At its conclusion, this project is expected to (Aim 1) demonstrate that DsbA is required for post- translational disulfide bond formation in EntV, and is also required for E. faecalis virulence (Aim 2). Understanding the contribution DsbA, and the novel observation of its requirement for pathogenicity will inform the future development of new therapeutics effective against C. albicans and E. faecalis.
Disulfide bond formation catalyzed by the Enterococcus faecalis thioredoxin DsbA has never been studied and is generally thought to play only a minor role in Firmicute protein maturation. This proposal will examine the contribution of DsbA towards the post-translational disulfide bond formation of the bacteriocin EntV, and in additional DsbA substrates required for virulence. Findings identified by this study may indicate novel targets for therapeutic intervention. !
