New Scholar Award in Global Infectious Disease
Massachusetts General Hospital
Use of the Caenorhabditis elegans model of fungal pathogenesis to identify virulence factors of the pathogenic yeast Cryptococcus neoformans
The significant numbers of serious fungal infections, the paucity of new antifungal agents, and the likelihood of the emergence of drug resistance in fungi, suggest a pressing need for new model systems to study the mechanisms of fungal virulence and host response. Especially cryptococcal meningitis is a relatively common and often fatal complication of HIV disease with a mortality that in developing countries can exceed 40%. However, mammalian models used to study fungal infections are cumbersome and expensive and using a mammalian host to screen cryptococcal mutant libraries for avirulent mutants is prohibitively time consuming and expensive.
Non-vertebrate hosts can be used to identify novel virulence determinants in a variety of microbial animal pathogens. A variety of bacterial pathogens of humans also kill Caenorhabditis elegans, that this killing is dependent on genes involved in mammalian infection, and that the C. elegans model can be used to identify new virulence factors. Recently, we expanded the use of the C. elegans model of microbial pathogenesis, to study the pathogenesis of the model human pathogen yeast Cryptococcus neoformans. We found that the C. neoformans polysaccharide capsule, as well as several C. neoformans genes previously shown to be involved in mammalian virulence were also shown to play a role in C. elegans killing. These included genes associated with signal transduction pathways (GPA1, PKA1, PKR1, and RAS1), laccase production (LAC1), and the alpha mating type. When we used a reporter gene of green fluorescent protein (GFP) fused to the mating pheromone MF1a, we observed GFP expression only in yeast cells within the nematode intestine, and not elsewhere, analogous to results obtained in a rabbit model of cryptococcal meningitis. In addition to the similarities in the virulence traits utilized by C. neoformans in both mammalian infection and C. elegans killing, the nematode response to cryptococcal infection also has some interesting similarities to that of mammals.
Using the C. elegans/C. neoformans model as a screen will allows us to focus on the mutants that have the best chance to be associated with pathogenesis and then confirm their role using a small number of mice. Results from evaluating random and targeted mutants of C. neoformans indicate that the C. elegans/C. neoformans model can provide useful insights into cryptococcal pathogenesis. For example, we have recently screened a set of 300 random mutants of C. neoformans H99. Fifteen (5%) of these mutants were attenuated on initial C. elegans screen and nine of these mutants (3%) were consistently attenuated on repeated evaluation with higher numbers of nematodes. This finding is similar to what we and others have found using large libraries of bacterial pathogens and suggests that the screen of a large library of random mutants of C. neoformans can provide useful insights in cryptococcal pathogenesis.
It is anticipated that the proposed investigations will advance our understanding of the pathogenesis of this significant human fungal pathogen, provide possible targets for the development of new antifungal agents, and contribute new insights on basic, evolutionarily preserved mechanisms by which yeasts interact with hosts during the pathogenesis process.