Prevention of invasive fungal disease in fish

Objective 1: Identify molecular mechanisms that mediate fish innate immune interactions with fungi.The overarching scope of our research program addresses questions that focus on how fish protect themselves against fungal pathogens. This work includes discovery of mechanisms of infection tissue-to-bloodstream spread, fungal immune recognition by C-type lectins, and fungal-bacterial interactions.We primarily use zebrafish models of candidiasis to discover how fungi and host immune cells interact during infection. The transparency of larval zebrafish enables unparalleled study of pathogen dissemination, and we use a yolk infection route to identify host and pathogen components that regulate this important event in establishment of infection. The yolk infection route is especially powerful to dissect processes of infection spread because it is a localized infection site that permits dissemination from tissue into blood. We are currently perturbing the host by limiting phagocyte function to determine their roles in limiting infection and dissemination. This is being done using 1) macrophage ablation through conditional ablation with metronidazole, 2) macrophage ablation with clodronate liposomes, 3) morpholino- mediated blockade of myeloid development to eliminate macrophages and neutrophils, and 4) creating a transgenic zebrafish line that will allow conditional neutrophil ablation with metronidazole. We are also manipulating the fungus genetically to determine the contributions of different morphologically specialized growth types to dissemination and tissue invasion. We are also characterizing immune responses of zebrafish to swimbladder infections with different Candida species in order to discover species-specific infection strategies. Finally, we are using the zebrafish to probe the role of immune response in the outcome of mixed bacterial-fungal infections. We have chosen bacteria that normally co-habitate niches in the human, including oral bacteria and bacteria co-isolated with C. albicans from the lung.These diverse studies are designed to provide unique insights both into mechanisms of fungal pathogenesis and into zebrafish immune strategies. As a whole, they leverage our ability to directly image fungal and immune cells during infection progression. As we have already shown using this model, these methodologies can reveal new fungal and host components that are important for spatiotemporal regulation of infection.Objective 2: Determine how neutrophils prevent invasive mucosal disease in fish.Preliminary results suggest neutrophils are important for immunity to mucosal candidiasis in the swimbladder, and that extracellular traps are produced during infection. Specifically, blockade of neutrophil recruitment by overexpression of a dominant-negative allele of Rac2 increases infection intensity and mortality. Further, we find hyphae decorated with extracellular DNA, suggesting the presence of extracellular traps. Intriguingly, immunity is also compromised upon inhibition of the chemokine receptor CXCR2, which mediates neutrophil recruitment and activation. Our working hypothesis is that mucosal fungal containment and extracellular trap formation is driven by recruitment of neutrophils and chemokine signaling including CXCR2. We will use reverse-genetics, chemical inhibition, and high-resolution imaging to identify mechanistic aspects of each of these aspects of neutrophil immune response. In addition to neutrophil involvement, preliminary results suggest there is a moderate macrophage interaction with C. albicans in the infected swimbladder. We will further characterize this response and determine if macrophages play a protective role against mucosal candidiasis in the fish.