Responses to Antifungal and Alkaline pH Stress in Human Fungal Pathogens

Abstract

The fungal pathogens encounter ever-changing conditions during their pathogenic life cycles, including shifts from the environment to the human host. Fungi have evolved many pathways that allow them to overcome and even thrive in the presence of the stresses presented by different environments.Antifungal drug treatment presents a significant stress for the opportunistic pathogen Cryptococcus neoformans. Currently, there are limited treatment options for cryptococcal infections. Researchers have been working toward identifying drugs that inhibit fungal-specific processes, such as the echinocandins, which target the synthesis of the fungal cell wall component β-1,3-glucan. However, C. neoformans is highly tolerant of these drugs, despite their effective inhibition of the sole and essential cryptococcal β-1,3-glucan synthase. We therefore performed a screen through a deletion mutant collection to identify compensatory processes involved with echinocandin tolerance. In this way, we identified several processes that are required for full tolerance of these drugs, including stress-induced responses and cell wall biosynthesis. Overall, these studies implicate distinct and targetable cellular processes that might be exploited to enhance echinocandin efficacy for treating infections caused by C. neoformans.An alteration in extracellular pH is another one of the predictable changes that C. neoformans encounters in its shift from its environmental niche to the human host. This environmental increase in pH is internalized into microbial cells through the fungal-specific Rim signal transduction pathway. We have determined that the upstream pH-sensing components of Rim signaling are significantly divergent in the basidiomycete phylum, which includes C. neoformans, agricultural pathogens, and saprophytes. Recently, we identified the first basidiomycete Rim pathway pH sensor, the C. neoformans Rra1 protein. Through a proteomics-based screen for potential Rra1 pH sensor signaling partners or interactors, we identified nucleosome assembly protein 1 (Nap1) as an interactor of the Rra1 pH sensor. Like Rra1, C. neoformans Nap1 is required for the activation of the Rim pathway. Nap1 specifically interacts with the Rra1 protein, acting as a scaffold to maintain stability of the Rra1 pH sensor protein in the cell. In current work, we are exploring how Nap1 and other proteins regulate the fungal pH sensing complex, through both localization and post-translational modification of the Rra1 pH sensing protein.Finally, I expanded these studies into another basidiomycete fungus, the skin commensal and pathogen, Malassezia sympodialis. In this fungus, I confirmed that the Rra1 pH sensor is, in fact, a basidiomycete-specific protein and that a functional Rim pathway is required for growth at high pH or salt concentrations. Finally, through RNA-sequencing analyses, we identified genes that are regulated by the Rim pathway in response to alkaline pH. These studies have expanded our knowledge about Rim pathway function in basidiomycete fungi.Together these inter-related experimental approaches explore ways in which C. neoformans adapts to overcome and survive stressful environments. We have identified novel signaling elements of conserved, stress-response pathways in fungi. Additionally, we have explored mechanisms by which important human pathogens display intrinsic tolerance to established antifungal agents, providing insight into potential new avenues for antimicrobial therapy.