Browsing by Subject "Fungal pathogen"

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.

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.

Microorganisms must regulate genomic stability to strike a balance between excessive deleterious mutation and evolutionary stagnation to successfully compete and endure within their ecological niches. Two important mechanisms involved in maintaining genomic stability are RNA interference (RNAi) and DNA mismatch repair (MMR). RNAi defends the host genome by targeting double-stranded viral RNAs and aberrant endogenous RNAs for degradation. Endogenous sources of aberrant RNAs include transcripts derived from transposable elements and repetitive sequences as well as transcripts with inefficiently spliced introns. Transcription and translation of these endogenous aberrant RNAs is often considered deleterious to the host because transposable elements are capable of replicating and spreading throughout the genome, a process that can disrupt genes and destabilize chromosomes. The DNA MMR pathway canonically detects mismatches caused by DNA damage or errors during DNA replication. After recognizing mismatches, MMR pathway components recruit the appropriate proteins for removal and repair of the mismatched nucleotide. In addition to this role, MMR pathway components are also involved in the rejection of homeologous, or only partially homologous, meiotic recombination intermediates. This activity mediates a critical role in the maintenance of species boundaries, by inhibiting successful recombination between the genomes of two sufficiently divergent organisms, often preventing the production of viable or fertile progeny.The first chapter of this dissertation begins by introducing pathogenic Cryptococcus species, their ability to mediate disease in humans, and various aspects of their genomes and life cycles. Following the introduction to Cryptococcus, factors known to mediate genomic instability in fungi are described. In Chapter 2, the identification and characterization of two clinical Cryptococcus neoformans isolates with significantly increased mutation rates due to RNAi loss and rampant mobilization of a transposable element are detailed. Chapter 3 describes the impact of loss of a functional MMR pathway on the species boundary between C. neoformans and Cryptococcus deneoformans, sister species within the pathogenic Cryptococcus species complex. In Chapter 4, experimental procedures for conducting genetic crosses with Cryptococcus, isolating meiotic products, and many factors impacting these methods are presented. The conclusions of each preceding chapter are then summarized in Chapter 5, where I also put forth further questions and directions for each project. In Appendices A and B, two ongoing projects focused on the identification of additional RNAi-deficient C. neoformans strains as well as work to discover novel RNAi components are respectively described. Lastly, Appendices C and D include supplementary tables from Chapters 2 and 3, respectively.