Author ORCID Identifier

0000-0002-2761-7225

Date of Graduation

12-2017

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Danielle A. Garsin

Committee Member

Michael C. Lorenz

Committee Member

Ambro van Hoof

Committee Member

Barrett R. Harvey

Committee Member

Michael C. Gustin

Abstract

The human microbiome is a diverse polymicrobial population comprised of both fungi and bacteria. Perturbations of the normal microbiome can have a profound impact on health, including the development of infections. Exploitation of these polymicrobial interactions has the potential to provide novel treatment and prevention strategies for infectious diseases. Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species are also amongst the most important and problematic, opportunistic nosocomial pathogens and are often co-isolated during infection. Surprisingly, these two species antagonize each other’s virulence in both nematode infection and in vitro biofilm growth.

Herein we identify the E. faecalis secreted bacteriocin, EntV, as both necessary and sufficient for the reduction of C. albicans virulence and biofilms through the inhibition of hyphal formation, a critical virulence trait of C. albicans. Furthermore, we demonstrate that the EntV propeptide is proteolytically processed by the metaloprotease, GelE, in E. faecalis, resulting in production of a highly active peptide of 68-amino acids, EntV68. The mature peptide effectively blocks biofilm development in multiple media conditions and disrupts pre-formed biofilms, which are resistant to current antifungal treatments. Moreover, EntV68 is efficacious against clinical strains of C. albicans, including azole resistant strains. Biofilm development of other pathogenic Candida species is also impeded by EntV68 treatment, demonstrating the target spectrum of EntV68 is not limited to C. albicans. EntV68 is protective in the murine macrophage and oropharyngeal candidiasis (OPC) infection models at nanomolar concentrations. Epithelial invasion, inflammation, and fungal burden in the OPC model were significantly reduced in response to treatment with EntV68. Collectively, C. albicans cells present in the hyphal form were greatly reduces in all models examined. Despite these profound effects, EntV68 has no effect on C. albicans viability, even in the presence of significant host-mimicking stresses. EntV68 associates with cell surface of both yeast and hyphal cells of C. albicans and deletion of genes involved in hyphal morphogenesis and cell wall composition abrogate the inhibitory activity in C. albicans biofilms. These findings demonstrate that EntV has potential as a novel antifungal agent that targets virulence rather than viability.

Keywords

Candida albicans, Enterococcus faecalis, polymicrobial interactions, commensalism, pathogenesis, bacteriocins

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