No abstract

Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited treatments. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells, necessitating long treatments. To improve treatment, we used our novel high-throughput method, the overlap2 method (O2M) to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

SummaryInvasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited number of efficacious drugs and poor drug availability. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits fungal cell growth instead of killing fungal cells, necessitating long treatments. To improve fluconazole treatments, we used our novel high-throughput method, the overlap2 method (O2M), to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. Although serendipitous identification of these interactions is rare, O2M allows us to screen molecules five times faster than testing combinations individually and greatly enriches for interactors. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with disseminated Cryptococcus neoformans infections. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

SUMMARYPhenotypic heterogeneity is a common microbial strategy to improves fitness in fluctuating environments. The fungal pathogen Cryptococcus neoformans exhibits dramatic size heterogeneity: it varies from 2-100 μm in diameter during mammalian infection. Following pulmonary invasion, cells enlarge to >30 μm diameter, then decrease over disease course. Extrapulmonary organs, particularly the brain, contain uniformly small cells, implying that that morphotype is important for dissemination. To test this hypothesis, we isolated size-based ex vivo cell populations directly from mouse lungs. Small ex vivo cells readily disseminated compared with other ex vivo populations, small beads, and in vitro-grown small cells. The latter two groups are close in size to small ex vivo cells, suggesting that while size is important, fungal-specific elements also drive extrapulmonary dissemination. We found that mannose exposure facilitates host cell interaction and organ uptake. Phosphate induces small cell formation. This demonstrates how environmental cues shift phenotypic heterogeneity to drive pathogenesis.

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