Browsing by Subject "RNAi"

Dengue fever and dengue hemorrhagic fever are estimated to afflict 50-100 million people annually and are caused by one of the four serotypes of dengue virus. Dengue virus is carried and transmitted to humans by mosquitoes of the Aedes genus. Given the broad geographic distribution of Aedes mosquitoes, it has been estimated that nearly half the world's population is at risk of contracting the disease. Currently, no vaccine or specific antiviral treatment is available to combat this emerging menace.

A greater understanding of how dengue virus interacts with its insect and human hosts will facilitate the intelligent design of specific antivirals to combat the disease and enable the selective breeding of mosquitoes resistant to the virus. Although the genomes of the two primary mosquito vectors have been sequenced, the molecular tools necessary for conducting a systematic genetic analysis of host factors required for DEN infection are not yet available. These tools do however exist in the closely related fruit fly, Drosophila melanogaster. By using a strain of dengue virus that was adapted to propagate in fruit fly cells, we completed a full genetic screen for host factors required for efficient dengue virus propagation. When homologues of these host factors were assayed in a human cell line, over half were also shown to be required for efficient viral propagation. This indicates that while the virus is utilizing many of the same pathways in both of its hosts, the interaction with the insect vector has unique features that may contribute to the observed lack of pathogenesis in mosquitoes.

Nociceptors are specialized primary sensory neurons that represent the first line of defense against potentially tissue damaging environmental stimuli, and are involved in pathological pain states caused by nerve damage, inflammation and many chronic diseases. In nociception, these neurons detect harmful stimuli and contribute to the reactions to avoid them. Nociceptors transduce noxious stimuli into membrane depolarization, which in turn, triggers action potentials. These action potentials are conducted to synapses in the central nervous system (CNS), resulting in release of neurotransmitters at the presynaptic terminal. The unifying factor in the progression of nociceptive signaling i.e. transduction, action potential propagation, and neurotransmitter release, is the contribution of ion channels.

In this study, I use Drosophila melanogaster larvae as a model system to study the contribution of ion channels to nociception. Larvae stimulated with a noxious thermal or mechanical stimulus perform a stereotyped and quantifiable escape behavior. Larvae exhibiting this nocifensive behavior rotate around their long body axis in a corkscrew-like manner thus escaping the damage of the noxious stimulus. This behavior is triggered by the Class IV multidendritic (md) neurons, which are the main larval nociceptors. I describe here, the results of my systematic screen for ion channels required for larval thermal nociception. To perform this screen, I utilized RNAi to knock down the expression of 98% of the predicted ion channels in the Drosophila genome. I observed the effects of ion channel knockdown in the thermal nociception behavioral assay.

In addition, I present detailed characterization of an ion channel that I found to be critical for inhibition of nociceptor excitability, the small conductance calcium-activated potassium channel, SK. This channel inhibits both thermal and mechanical nociception. Results of calcium imaging studies show enhanced excitability of larval nociceptors in SK mutant animals. My findings support a role for SK function at the sensory afferents, cell body, and axon.

Another candidate ion channel gene, shadrach, encodes a Degenerin/Epithelial Na+ channel (DEG/ENaC) that I found to be required for thermal nociception. DEG/ENaCs are conserved in flies, nematodes, and several vertebrates including humans. These channels are expressed in a variety of tissues including kidney epithelia, muscle, and neurons. Members of this superfamily play a role in a host of biological processes including salt homeostasis, neurodegeneration, proprioception, touch transduction, and nociception. RNAi knockdown of shadrach results in increased thermal nociceptive threshold. Optogenetic experiments suggest that shadrach functions downstream of transduction.

Furthermore, I identified seven ion channel genes in the thermal nociception screen, which affect nociceptor dendrite morphology. It is possible that thermal nociception behavioral phenotypes in these RNAi mutants are a consequence of the altered dendritic field. Reduction in segmental coverage by the nociceptors may influence the ability to detect noxious stimuli. Future research in our laboratory will establish the relationship between these ion channels, nociceptor development, and nociceptive behavioral output.

Drosophila melanogaster is emerging as a powerful model for the study of pain signaling. I have uncovered several candidate ion channel genes that contribute to thermal nociception; of these, SK and shadrach are required for the response to noxious heat. I have shown that dendritic field coverage is important for the detection of noxious stimuli, and I have identified many candidate genes that are required for normal dendrite morphology.

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.

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.

Sexual development enables microbial pathogens to purge deleterious mutations from the genome and drives genetic diversity in the population. Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. C. neoformans has a defined a–α opposite sexual cycle (bisexual reproduction); however, >99% of clinical and environmental isolates are of the α mating type. Interestingly, α cells can undergo α–α unisexual reproduction, even involving genotypically identical cells. A central question is why would cells mate with themselves given that sex is costly and typically serves to admix pre-existing genetic diversity from genetically divergent parents? Sexual reproduction generates abundant spores that following inhalation, they penetrate deep into the alveoli of the lung, germinate, and establish a pulmonary infection growing as budding yeast. Therefore sex has been linked with virulence; however, hyphal development has been previously associated with reduced virulence and thus the roles of morphogenesis in virulence have not been extensively analyzed. To further understand the role of unisexual reproduction in C. neoformans we will investigate the evolutionary implications of α–α mating, explore its role in pathogenesis, and we will dissect the signaling pathway that regulates sexual development.

We isolated α–α unisexual reproduction progeny from the hyperfilamentous strain XL280 and subjected to a variety of phenotypic and genotypic assays (including whole genome sequencing and CGH). We found that unisexual and bisexual reproduction frequently generates phenotypic and genotypic diversity de novo, including aneuploidy. Aneuploidy was responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments, such as the mammalian host.

Interestingly aneuploidy strains that were fluconazole resistant were as virulent as the WT parental strain XL280. Although XL280 belongs to the serotype D lineage that exhibits limited pathogenicity, in further studies we found that is hypervirulent in the murine model. It can grow inside the lung of the host, establishing a pulmonary infection, and then disseminates to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is characterized by less protective immunity and is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies: 1) provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans (serotype D), and 2) reveal the virulence potential of serotype D that is broader and more dynamic than previously appreciated.

Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. To address this question we applied an insertional mutagenesis via Agrobacterium tumefaciens transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 governs cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. In further studies we found that Znf3 is required for sex- and mitotic-induced (SIS and MIS). SIS is less efficient in znf3 unilateral matings and is abolished in znf3 x znf3 bilateral matings, similar to the phenotypes of rdp1 mutants (the RNA-dependent RNA-polymerase of RNAi pathway). Znf3 is also required for transgene-induced mitotic silencing; znf3 mutations abrogate silencing of repetitive transgenes during vegetative growth. Znf3 tagged with mCherry is localized in the cytoplasm in bright, distinct foci. Co-localization of Znf3 with the P-body marker Dcp1-GFP further supports the hypothesis that Znf3 is a novel element of the RNAi pathway and operates to defend the genome during sexual development and vegetative growth. In concussion our studies provide further understanding of unisexual reproduction as an evolutionary successful strategy.