Browsing by Author "Perfect, John R"

Invasive fungal infections cause significant worldwide morbidity, and mortality rates remain remarkably high despite treatment. One of the most pressing fungal pathogen threats is the opportunistic infection, Cryptococcus neoformans. C. neoformans is a yeast found ubiquitously throughout the environment that can cause cryptococcal meningitis, primarily in immune suppressed individuals. Treatments for C. neoformans and other invasive fungal pathogens have a number of disadvantages. Currently, there are only four classes of antifungals available for treatment, and each has varying efficacy and toxicity profiles. Given the overlap in eukaryotic cellular machinery between fungal pathogens and their human host, many of the most effective treatments have significant associated toxicities, side-effects, and drug interactions. Additionally, many antifungal drugs are unavailable or inaccessible to the patient populations in greatest need. Therefore, novel therapeutic strategies and antifungal targets are urgently needed to address the threat of invasive fungal infections. My thesis work has subsequently focused on investigating novel treatment strategies, potential druggable targets, and novel antifungal compounds to combat C. neoformans and other invasive fungal pathogens.Given the limited options available for the treatment of invasive fungal infections, we employed a creative strategy to evaluate the effect of diet on antifungal drug efficacy. We determined that a high-fat, low-carbohydrate, adequate protein ketogenic diet potentiated the antifungal effect of fluconazole in vivo against murine models of systemic C. neoformans and Candida albicans infection. Pharmacokinetic/pharmacodynamic analysis revealed that a ketogenic diet significantly enhanced drug exposure in both blood plasma and brain tissue. However, this pharmacokinetic enhancement was not exclusively responsible for observed effect on tissue fungal burden, indicating a yet-unknown mechanism of fluconazole activity enhancement. We further evaluated the effects of a ketogenic diet on the immune system, the effect of ketone bodies on C. neoformans, and the activity of other antifungal compounds and diets. While the mechanism remains elusive, our findings indicate that a ketogenic diet potentiates the activity of fluconazole against C. neoformans and C. albicans at multiple body sites of infection. These results may have promising practical treatment implications in the future. C. neoformans is characterized by a strong brain tropism that has largely confounded researchers, as the brain traditionally presents a hostile, nutrient poor, and oxidatively stressful environment. However, C. neoformans is adept at establishing infection at this site. We asked whether nitrogen metabolism may play a key role in C. neoformans infection, especially within the central nervous system. Through RNA-seq analysis from Cryptococcus isolates derived directly from patient cerebrospinal fluid, we identified that genes encoding enzymes surrounding glutamine and glutamate metabolism were highly upregulated during infection. In particular, glutamine synthetase was found to be one of the most highly expressed genes of the entire Cryptococcus genome during central nervous system infection. We subsequently investigated the plasticity of this central nitrogen hub that interconverts glutamate and glutamine through the construction of multiple gene deletion mutations of its core enzymes. Surprisingly, while C. neoformans could tolerate loss of multiple enzymes in this central nitrogen hub, we determined that the glutamine synthetase enzyme is likely essential. Further work utilizing an inducible promoter system and known glutamine synthetase inhibitors supported this hypothesis. We finally evaluated a toxic analog of glutamine—the enzymatic product of glutamine synthetase, in an in vivo model of systemic C. neoformans infection. Taken together, we determined that glutamine synthetase is essential in C. neoformans, and glutamine metabolism may serve as a promising and novel druggable target for future antifungal drug development. I have next presented an overview of the opportunities for repurposing anticancer drugs to treat fungal infections and introduce two novel antifungal compounds currently in preclinical development. I have additionally provided a summary of findings presented in this dissertation, as well as several avenues for future research. Collectively, my thesis work has highlighted several novel strategies, targets, and compounds for future antifungal therapeutic development, and these studies emphasize the need for creativity and interdisciplinary thinking in the development of antifungal therapies.  

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH.

Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms.

The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi.

To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.

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