Opportunistic infections in hospitalized patients remain an important unsolved clinical problem, leading to an estimated 230,000 annual deaths in the US and Europe. Two of the most important opportunistic pathogens are the fungus Candida albicans and the gram-negative bacterium Pseudomonas aeruginosa. They are frequently co-isolated in ventilated patients and in cystic fibrosis, where co-association is linked to exacerbated disease. Drug treatment of these two opportunistic infections is often problematic, and we hypothesize that some of these failures are due to the impact of microbe-microbe and microbe-immune interactions during co- infection. The proposed research seeks to address this major gap in our understanding to define whether the complexity of co-infection demands a different view of therapeutic strategy. Our overall goal is to understand how Pseudomonas-Candida interactions affect virulence and therapeutic success. We recently found that the antifungal drug fluconazole (FLC) is much more effective and fungicidal against C. albicans in the presence of P. aeruginosa, both in vitro and in vivo. In this proposal, our driving hypothesis is that specific bacterial virulence factors block fungal tolerance to FLC. We hypothesize that iron starvation and calcineurin signaling are two key elements in this synergy, based on our preliminary data as well as published work connecting iron homeostasis and calcineurin to drug tolerance. Leveraging the power of intravital imaging in zebrafish together with microbial genomics will enable our genetic analyses of bacteria and fungi in the context of treatment and infection. We propose to uncover novel bacterial and fungal contributors to P. aeruginosa-FLC synergy against C. albicans through a complementary candidate gene/unbiased screening strategy in vitro (Aim 1). We will then utilize longitudinal intravital imaging of fungi, bacteria, and innate immune cells in the transparent zebrafish to test the relevance of these mechanisms and the innate immune contributions to fungal therapy during co-infection in the vertebrate host (Aim 2). The findings from these proposed studies will yield a network of bacterial and fungal pathways that operate in vivo to regulate drug susceptibility. This proposal has the potential to open up a new front in our study of how communication between bacteria and fungi during infection regulates drug susceptibility. While this is a basic science research proposal that will reveal new cross-kingdom interactions during infection, the focus on antifungal treatment also has significant potential for informing future translational research.

Infectious diseases affect humans worldwide and cause significant mortality and morbidity in the United States. Current methods to control fungal disease are inadequate, especially in the immunocompromised patient population, so it is critical to investigate alternative treatments. A long-term goal of this investigation involves leveraging basic information about host-pathogen interaction to develop therapeutics to treat fungal disease.