Investigation of key proteases in the parasitic phase of Coccidioides

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Project Summary Coccidioides spp. are major fungal pathogens endemic to the Southwest United States, Central America, and South America. In recent years, the incidence of coccidioidomycosis has continued to rise. Coccidioides infects and kills immunocompetent individuals when they inhale spores from the soil and, despite current antifungal treatment, continues to cause unacceptably high morbidity and mortality. The ability of Coccidioides to cause disease depends on an elaborate developmental transition from mycelia found in the soil to the host form known as a spherule, a morphology unique to Coccidioides. While this developmental process can be recapitulated in the lab, the molecular determinants of spherulation remain poorly understood. Delineating which genes are required for spherulation will lead to identification of therapeutic targets and accelerate discovery of new antifungals for the treatment of coccidioidomycosis. We will focus this project on two families of secreted proteases, subtilases and deuterolysins, and assess the role they play in spherulation. These protease families have undergone evolutionary expansion in the Coccidioides lineage and, since spherules are unique to Coccidioides, we hypothesize that their evolutionary expansion implicates them in the process of spherule development. Protease inhibitors have proven to be successful therapeutics for other infectious diseases, including HIV. The Coccidioides proteases that we discover to be required for the process of spherulation will be excellent candidates for therapeutic targets. We will use a multipronged approach, including transcriptional profiling, protease inhibitor studies, and genetics, to elucidate the role of key proteases in spherulation. This will leverage PI Sil’s extensive experience with transcriptional profiling of dimorphic fungi, including Coccidioides, and collaborator Dr. Craik’s expertise on the biology of proteases, including previous work on secreted proteases in pathogenic fungi. In Aim 1, we will identify candidate secreted proteases involved in spherulation by profiling the transcriptome of multiple developmental stages of Coccidioides to identify proteases whose expression is spherule-enriched. Additionally, we will determine the effect of class-wide protease inhibitors on spherule formation, thereby implicating individual protease families as key players in Coccidioides spherule development. In Aim 2, we will generate six mutant Coccidioides strains, each lacking a secreted protease which is already implicated in spherulation from our preliminary studies. Additionally, we will generate six more protease mutants based on prioritization from studies in Aim 1. Using these deletion mutants, we will test whether each protease is required for spherulation. Our work will provide a rich transcriptional profiling dataset as well as multiple deletion mutants that are critical for dissecting the role of these two expanded protease families in the parasitic phase of Coccidioides biology.