Browsing by Subject "Cryptococcus"

One 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.

Fungi of the basidiomycete genus Cryptococcus cause disease in an estimated quarter of a million people, annually. Cryptococcus neoformans and Cryptococcus deneoformans are the two most prevalent disease causing species within the Cryptococcus clade, with isolates of these species exhibiting considerable variation in their pathogenicity, ranging from benign to highly virulent. A wide variety of traits, such as thermal tolerance, melanin production, and an extracellular capsule contribute to virulence, yet our understanding of the genetic architecture of such traits is limited. In the studies reported here, I describe the first genome-wide analyses of recombination in C. neoformans and C. deneoformans and provide the first high-resolution genetic mapping studies of virulence traits in these important fungal pathogens.

In studying recombination, I considered both the nuclear and mitochondrial genomes, and estimated recombination rates for both opposite- and same-sex matings. With respect to recombination of the nuclear genome, I found that progeny from opposite-sex mating have more crossovers on average than those from same-sex mating. These analyses also suggest differences in recombination rate between C. neoformans and C. deneoformans. Similarly, analyses of mitochondrial inheritance and recombination point to differences between offspring from opposite- and same-sex matings, though with much lower overall rates of recombination as compared to the nuclear genome.

To dissect the genetic architecture of complex virulence traits, I employed quantitative trait locus (QTL) mapping. A unique aspect of these QTL studies was the application of functional data analysis methods that exploit time-series data and multiple experimental conditions. I mapped QTL for thermal tolerance, melanization, capsule size, salt tolerance, and antifungal drug susceptibility in C. deneoformans. For several QTL, I was able to identify candidate causal variants that underlie these loci. Two major effect QTL for amphotericin B resistance map to SSK1 and SSK2; regulators of the high osmolarity glycerol (HOG) pathway that governs responses to osmotic stress. Epistatic interactions between SSK1 and SSK2 were also shown to govern fludioxonil sensitivity. A third major effect, pleiotropic QTL was mapped to the gene, RIC8, a regulator of cAMP-PKA signaling. RIC8 variation is predicted to contribute to differences in thermal tolerance, melanin production, and capsule size.

In combination, the studies reported here advance our understanding of the mechanisms that generate and maintain variation in Cryptococcus and implicate genetic variants in key stress-responsive signaling pathways as a major contributor to phenotypic variation between lineages of Cryptococcus.

Cryptococcus neoformans pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of C. neoformans yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.

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.

Fungi have a genetically controlled sex determination system, which is governed by a small, sex-specific region in the genome called the mating-type locus (MAT). In the basidiomycetous yeast Cryptococcus neoformans, the pathogen that causes cryptococcal meningitis and cryptococcosis, sex has been associated with virulence. To further understand how sex is genetically regulated in C. neoformans, we focused our studies on the evolution of the MAT locus and molecular dissection of the pheromone signaling pathway that controls sexual development. Two MAT-linked meiotic recombination hotspots that likely drove the assembly and rearrangement of MAT were identified. Fine mapping through the integration of genetic markers established that two hotspots, one on each side of the MAT locus, are located in an ~10 kb and ~5 kb region. Plotting the G + C content along MAT and the flanking regions revealed a strong association between the location of these two hotspots and a high G + C content. By deletion and insertion of the G + C rich region, we demonstrated that the high G + C rich region is required but not sufficient to induce recombination. On the other hand, to provide direct experimental evidence to support the previously proposed model for the evolution of MAT, we sought to recapitulate the ancestral tetrapolar, and the intermediate tripolar mating systems of C. neoformans by manipulating the MAT structure to model a tetrapolar system. In the two modified "a" and "α" strains, the sex-determining genes SXI1α or SXI2a residing at the MAT locus were disrupted and the wild-type allele of these two genes was then reintroduced at another genomic location (URA5) that is unlinked to MAT. Our results show that C. neoformans can complete the sexual cycle with a tetrapolar mating configuration and the transitional tripolar state might be under strong negative selection pressure, which could have facilitated the transition from a tripolar state to the final bipolar mating system.

The MAT locus is the major determinant of the sexual identity of a cell, but several signaling pathways, including the pheromone signaling pathway, are required to regulate mating and sexual development. Many components of the pheromone signaling pathway have been identified; however, it is less clear what lies upstream of the MAPK cascade. To address this question, we studied the role of two Gα subunits (Gpa2, Gpa3) in mating and concluded that they share both redundant and divergent roles in mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. In their GTP-bound form, they signal in opposition: Gpa2 promotes mating whereas Gpa3 inhibits. Furthermore, we also studied the functions of a novel upstream component Cpr2, a pheromone receptor-like gene, in pheromone signaling and sexual development. All lines of evidence suggest that Cpr2 is a constitutive ligand-independent receptor that, when expressed, engages the same G-proteins and activates the same pheromone signaling pathway as the canonical ligand-activated pheromone receptors. Expression of Cpr2 is induced post cell fusion during mating, and likely introduces a positive feedback loop to allow a self-perpetuating signaling state to enable efficient mating. Cells lacking this receptor are fertile, but produce abnormal filamentous structures. Overexpression of CPR2 in a or α cells strongly enhances fruiting, an alternative same-sex mating process in C. neoformans. Therefore, Cpr2 establishes a new paradigm for a naturally occurring constitutively active GPCR that governs cell fate in fungi.

Finally, we described a sex-induced silencing (SIS) phenomenon in C. neoformans. Using genetic approaches, we showed that SIS is triggered by a tandem insertion of a transgene during the sexual cycle. Interestingly, only a proportion of progeny carrying the transgene are silenced. Gene deletion, RIP, or DNA methylation do not contribute to SIS but the RNAi machinery is required. In conclusion, these studies provide further understanding of sex in C. neoformans from different perspectives, which invites comparisons to other fungal and even more broadly, eukaryotic pathogens to address the role of sex in evolution.

The evolution of pathogenesis, in many cases, is a story of competition between host and microbe; however, many opportunistic pathogens are primarily found in niches other than the host environment. Such pathogens frequently lack host-to-host transmission, and there may be limited opportunities for an infectious population to be re-dispersed back into the environment. Observations such as these motivate the hypothesis that the evolution of virulence traits in opportunistic pathogens may be primarily driven by environmental selective pressures, rather than the host-environment per se.

For Cryptococcus the ability to survive interactions with macrophages and the ability to grow at host body temperatures are indispensable to its pathogenic capabilities. The work presented here aims to dissect the genetic underpinnings of these virulence traits using the abundant natural variation of Cryptococcus and using the accumulation de novo mutations associated with growth under relevant stressors.

An important aspect of the hypotheses surrounding Cryptococcus evolution is the predator-prey interactions it has with free-living amoeba. Amoebae are able to consume Cryptococcus cells in a manner similar to how macrophages phagocytose and digest infectious cells. This similarity is the basis of the “Amoeboid Predator-Fungal Animal Virulence Hypothesis” which posits that amoeba act as training grounds for environmental fungal pathogens and thus inadvertently select for resistance to immune phagocytes. I tested this hypothesis by using QTL mapping to identify genes and alleles that are involved in amoebae resistance in both C. neoformans and C. deneoformans. I identified QTL that contribute to amoeba resistance, and discovered that the largest effect QTL in both species localize to homologous regions of the genome, suggesting a shared mechanism of amoeba resistance. In C. neoformans, this QTL also contributes to variation in melanization. I identified a causal variant for this QTL, a non-coding deletion upstream of a transcription factor, BZP4. Contrary to the predictions of the Amoeboid Predator-Fungal Animal Virulence Hypothesis, I did not find an association between the ability to survive amoeba predation and virulence in either in vitro or in vivo models of infection. These findings suggest a re-evaluation of the amoeba predation model for the evolution of pathogenesis, suggesting that factors other than amoeba may provide the significant selective pressures that underlie virulence ability.

I extended my quantitative analyses of Cryptococcus to two important factors involved in both environmental and disease contexts: thermal and low pH tolerance. In doing so, I discovered multiple pleiotropic QTL involved in general growth that also dictate stress tolerance in both high temperature and low pH environments. By fitting growth data to a Gompertz growth model and QTL mapping based on the parameters of this model, I discovered a novel QTL that effects lag, the time it takes for a population of cells to begin growing at an exponential rate. This lag QTL is pleiotropic across growth conditions. I identified a candidate allele for the lag QTL, a 9-bp deletion in CNAG_01111, a gene that has been found to impact growth initiation in other species of fungi.

Finally, taking a complimentary approach to understanding the role of genes in environmental survival, I experimentally evolved a C. neoformans strain in conditions of thermal stress and fludioxonil stress. I discovered that strains evolved at high temperatures lose tolerance to fludioxonil and strains evolved in fludioxonil lose temperature tolerance. Furthermore, the loss of fludioxonil tolerance in the high temperature evolved strains can be partially rescued by growing them on media containing fludioxonil. This rescue results in a proportional loss of thermal tolerance. Studying the genomic changes behind the evolved phenotypes I discovered multiple large scale deletions and one multi-gene duplication associated with fludioxonil resistance and a single multi-gene deletion associated with thermal tolerance. There are also a variety of small scale mutations associated with each evolved condition, including mutations of genes in the HOG and ergosterol pathway that are responsible for fludioxonil resistance. Mutations in uncharacterized multidrug transporters are frequently associated with fludioxonil resistance, suggesting that the evolved strains might also have altered resistance to other antifungals. These findings highlight the polygenic and pleiotropic genetic architecture of adaptation in C. neoformans on an ever warming planet with increased use of agricultural antifungals. The trade-offs found may represent a good sign for the use of phenylpyrroles as an agricultural antifungal.

Collectively, my work sheds light on genes and alleles involved in environmental survival while also making important connections back to human disease. It also exhibits the importance of utilizing the natural variation of fungal pathogens to study the evolutionary hypothesis surrounding virulence traits. The studies reported here also provide significant groundwork for many new insights into virulence genes and the origins of Cryptococcus pathogenicity.