Browsing by Subject "Hypermutation"
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Item Open Access Genome evolution in the fungal pathogen Cryptococcus deuterogattii(2017) Billmyre, Robert BlakeOne of the key challenges of the 21st century is the emergence and reemergence of pathogens. Fungal pathogens represent an important portion of this problem, as the cohort of immunocompromised patients susceptible to common fungal pathogens rapidly expands in the developed world, and HIV/AIDS continues to present substantial challenges in the developing world. Understanding the processes by which pathogens emerge is critical, both for treating current outbreaks and for preventing future outbreaks. Here I have focused on the human fungal pathogen Cryptococcus deuterogattii, an emerging pathogen responsible for an ongoing outbreak in the Pacific Northwest region of the United States and Canada over the past approximately 15 years. To do so, I have taken comparative genomic, population genomic, and classic genetic approaches to understanding the origin of the outbreak, and the evolution of virulence at three different levels. I will first compare C. deuterogattii to the rest of the Cryptococcus pathogenic species complex, followed by comparisons of clonal clusters within C. deuterogattii, and finally I will compare individual strains within the VGIIa cluster.
I will begin in Chapter 1 by introducing genetic and non-genetic drivers of phenotypic diversity. I will then introduce the diversity of the fungal RNAi pathway, as well as discuss the frequent losses of the RNAi pathway throughout the eukaryotic kingdom.
In Chapter 2, I will examine the differences between the C. deuterogattii species responsible for the Pacific Northwest outbreak and the non-outbreak members of the Cryptococcus pathogenic species complex. I began by comparing the R265 reference genome of C. deuterogattii to the C. neoformans, C. deneoformans, and C. gattii reference genomes. Here we discovered that the core components of the RNAi pathway, including both argonautes, one of the two Dicers, and the only RNA-dependent RNA polymerase had been lost in the C. deuterogattii genome. These gene losses are conserved across the entire species and are defining characteristics of the species. We utilized this information to conduct a comparative genomics screen and identified a total of 14 conserved genes that were lost in the C. deuterogattii genome. We tested these for function in the RNAi pathway and discovered that a number of previously uncharacterized genes are novel RNAi components. In total, 9 of the 14 genes have been shown to play a role in the RNAi pathway. We demonstrated that the sex-induced (SIS) and mitotic-induced (MIS) silencing pathways share core components, and that SIS requires additional pathway components not required by MIS, suggesting that SIS may be a related and more specialized version of MIS. Finally, we showed that SIS appears to be induced through the pheromone signaling and MAP kinase cascade.
In Chapter 3, I will describe a whole genome resequencing project where we sequenced and analyzed clonal outbreak strains from the Pacific Northwest, related strains from outside the outbreak, as well as representatives of diverse global isolates. We utilized phylogenomic inference to provide evidence that the three clonal subgroups of the outbreak had distinct proximal origins: VGIIa in South America, VGIIb in Australia, and VGIIc with no identified origin outside the United States. We also demonstrated that the C. deuterogattii population shows patterns consistent with ancestral mating, but shows little evidence of more recent mating events, meaning that the population is characterized primarily by long periods of clonal growth with only intermittent episodes of sexual recombination.
In Chapter 4, I will examine variation within the VGIIa and neighboring VGIIa-like groups of C. deuterogattii uncovered through our resequencing study. We identified an msh2 nonsense allele ancestral to the VGIIa-like group. Here we demonstrate that this allele is responsible for a mutator phenotype that is particularly severe in genes containing homopolymer runs. Mutator strains are uncommon in eukaryotic microbes, and this lineage may represent a rare stable and successful environmental hypermutator lineage. However, I will also present evidence that the mutator state leads to high mutational burden and eventually loss of virulence, and argue that the mutator allele did not play a role in the expansion of the Pacific Northwest Outbreak in the VGIIa group.
In Chapter 5, I will conclude this thesis and provide some thought towards future directions that emerge from this work. Finally, in Appendices A and B I will discuss a pair of unfinished projects focusing on identifying novel mycoviruses in RNAi deficient lineages and the genetic basis of 5-FC resistance.
Item Open Access Roles of RNA interference and DNA mismatch repair in maintaining genomic integrity in Cryptococcus pathogens(2022) Priest, Shelby JordanMicroorganisms 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.