Development of a fungal virulence assay using Caenorhabditis elegans as a model host to identify mechanisms of host pathogen interactions.

Jain, Charu

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Development of a fungal virulence assay using Caenorhabditis elegans as a model host to identify mechanisms of host pathogen interactions.

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Candida albicans is an opportunistic pathogen, which is responsible for causing systemic infection in immunocompromised patients in hospital settings (nosocomial infections). 90% of these nosocomial fungal infections are caused by C. albicans, and the estimated annual cost of treating them exceeds $1 billion per year. Despite this expenditure, there is a high mortality rate of 50%. There are two main routes of infections, first a mucosal infection that can spread and invades deeper into the tissues and gets disseminated into the bloodstream. Second, more frequently seen in hospital settings, is when Candida cells dislodge from a biofilm that has formed on intravenous devices or catheters. Treatment of these diseases is difficult due to a dearth of antifungal drugs and new strategies are required to explore mechanisms used by Candida in causing infection. One way of approaching these significant scientific challenges is to identify virulence determinants and mechanisms, which apart from providing insightful knowledge of fungal pathogenesis, can also be used as targets for antifungal drug development. The innate immune responses in humans,which are important for defense against fungal infections, are conserved in Caenorhabditis elegans. In order to identify Candidavirulence factors, I have developed a C.elegans based pathogenesis assay, where nematodes were infected with fungi (both S. cerevisiae and C. albicans) and observed for disease phenotypes including death. This assay can be used to study several aspects of disease progression in worms from swelling (inflammation a bio-marker of xinfection) to colonization in the intestine, leading to intestinal distension and ultimately death of the host worm. The assay offers a fast and simple way of identifying unknown genes, which when established as a virulence determinant in the worm model, can be further studied in mammalian models. I demonstrate the utility of this assay in multiple ways. First as proof of principle using this assay I have identified a fungal mutant cap1, which is susceptible to reactive oxygen species (ROS), and fails to cause disease, except in bli-3mutant worms that carry a mutation in an oxidase gene and is responsible for the oxidative stress. Second, we screened a library of ~1200 C. albicans mutants, and identified 7 genes, 3 known (CMP1, IFF11 and SAP 8), validating the assay and 4 novel genes (orf19.1219, orf19.6713, DOT4 and ZCF15) that play a role in fungal infection. Third use of this assay is to test potential drugs in a high throughput fashion. Families of related compounds were identified through a screen of 30,000 compounds, for their ability as potential inhibitors of C. albicans adhesion to biological and inert surfaces. These compounds were further tested in this assay for their ability to reduce infection of C. albicans in worms. The assay provides us with a method to test efficacy of antifungals in vivo. Finally, using the survival assay, a test for mortality caused by infection, we can observe disparity in the different C. albicans fluconazole resistant strains isolated from AIDS patients. In addition this assay after small modification can be potentially employed to screen the C. elegans RNAi library to identify the modulators of innate immune responses during fungal infection.

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