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Deepa Srikanta

Washington University in St. Louis
Molecular Microbiology
Insights into host:cryptococcal interactions using high throughput methods and RNAi
Cryptococcus neoformans, the causative agent of cryptococcosis, is an opportunistic fungal pathogen which kills over 600,000 individuals annually. Infection is through inhalation of the fungal particles. Once in the lungs, cryptococcal cells interact with host macrophages, the first line of defense against invading pathogens. When fungal cells adhere to host macrophages they can become engulfed, leading to a variety of outcomes that may benefit either the host or the invading pathogen. Cryptococcus neoformans has multiple virulence factors, several of which help it to evade the host immune system or replicate within host macrophages. Despite extensive research on cryptococcal pathogenesis, host genes involved in the initial uptake and subsequent stages of infection are woefully understudied. To rectify this situation we performed a high-throughput imaging screen where we exposed fungal cells to RNAi-treated host cells and evaluated uptake. Our initial screen of almost 1,000 host genes yielded 77 host factors that significantly affected cryptococcal uptake. Successive rounds of screening eliminated “hits” which could be attributed to general defects in phagocytosis and identified a group of fungal-specific host factors, several of which have never been implicated in fungal pathogenesis. Four of these genes significantly and specifically alter cryptococcal uptake and we chose two kinases (one involved in cell surface modification and another in cell signaling) for further validation based on access to primary mouse models and the intriguing function of these enzymes. Follow-up studies in primary cells (human and mouse) complemented the results of our initial screen and pilot studies in mice lacking the signaling protein show reduced fungal dissemination. Current studies are under way to determine the mechanism of action by which these proteins influence cryptococcal disease. Our exciting findings will shed light on host aspects of cryptococcal pathogenesis and potentially other host:microbe interactions as well.
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