Genetics - Theses
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ItemThe evolution of pathogenicity and isolate variation in Talaromyces marneffei( 2017)The opportunistic fungal pathogen of humans, Talaromyces marneffei, is one of very few pathogens in an order of over a thousand species and the only species that has the capacity to switch between two morphologically distinct growth forms (known as dimorphism). Growth at 25°C results in a saprophytic multicellular, hyphal form while infectious growth in a host occurs as a uninucleate unicellular yeast that resides within phagocytic cells of the immune system. The intracellular niche of T. marneffei differs significantly from the niches of other Talaromycetes. The identification of the mechanisms by which T. marneffei can survive and grow in this intracellular niche is a major aim of this study. Comparisons of the genomes of three closely related non-dimorphic, non-pathogenic species with the T. marneffei genome identified unique features that contribute to niche specific growth and the ability to cause disease. Most significant of these were an overall reduction in genome size and gene number in T. marneffei with substantial gene losses in families responsible for environmental interaction. These and other findings strongly indicate that T. marneffei has adapted to an intracellular host niche distinct from its saprophytic relatives. Against this background of gene loss three gene families were identified that had been significantly expanded in T. marneffei. These expanded gene families showed putative extracellular and cell surface localisation and consisted of cell wall galactomannoproteins (mpl family), aspartyl proteases (pop family) and a family of small proteins with very little functional characterisation in any species (mib family). Genes in the pop, mpl and mib families were over-represented in subtelomeric regions, under positive selection, had copy number variation in T. marneffei isolates and many had high levels of repetitive adjacent sequences including several transposon families. In the host T. marneffei grows as an intracellular pathogen within phagocytes and as such extracellular proteins interact directly with the host. Therefore another aim of this study was to characterise these expanded gene families and their role in pathogenesis. Deletion studies in pop genes revealed roles in yeast cell formation during intracellular growth, while high variability in cell-to-cell protein production for two mib genes suggested a role in cell surface variation when interacting with the host. Understanding the type and degree of variation within the population of a fungal pathogen can reveal its population structure and potential to adapt to stressors such as antifungal compounds. Genome wide variation in the T. marneffei population had yet to be examined therefore an aim of this study was to characterise the degree and type of this variation. To this end several clinical and environmental isolates of T. marneffei were examined for variation in chromosomal structure, which is a common means of generating phenotypic variation in other fungi. While no obvious abnormalities were observed, gene copy number variation in subtelomeric regions was widespread and several strains showed specific small mutations with impacts in antifungal resistance and phenotypic instability. Overall this study has revealed the genomic and genetic changes within T. marneffei and between it and other Talaromycetes. Many of these changes help to explain its unique niche as an intracellular pathogen within an almost entirely non-pathogenic clade. This research also highlights specific genes and gene families with roles in this pathogenesis and identifies potential therapeutic targets and genes involved in host interactions for future investigation.
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ItemIdentification and analysis of factors influencing intracellular growth of the pathogenic fungus Talaromyces marneffei( 2017)Fungal pathogens of animals and plants are a major concern in society with huge economic and public health consequences. The prevalence of pathogenic fungi and the emergence of new, opportunistic fungal pathogens, about which we know little, compounds the current problem. Talaromyces marneffei is a dimorphic, opportunistic pathogenic fungus that infects immunocompromised individuals. At 25 ̊C T. marneffei grows in a multinucleate hyphal form that can undergo a process of asexual development to produce conidia, which are the infectious agents. Upon inhalation, conidia reach the alveoli of the lungs where they are phagocytosed by resident phagocytes such as alveolar macrophages. The transition to 37 ̊C, which is the human host body temperature, induces the dimorphic switch to a pathogenic, uninucleate, fission yeast form. The yeast form is able to utilize macrophages as a niche from within which to avoid detection by the host immune system. T marneffei yeast must then be able to withstand macrophage-killing responses and obtain nutrients in order to proliferate inside these phagocytic cells. The objectives of this study were to determine the transcriptional response of T. marneffei to the host environment, including those induced by growth at body temperature as well as to host-derived cellular signals. This would lead to the identification of genes and pathways necessary for establishing infection. For this purpose RNAseq analysis was used to create a transcriptomic profile of T. marneffei during in vitro growth at 25°C (hyphal) and 37°C (yeast) and during murine and human macrophage infection (ex vivo). To identify pathways that are important during the establishment of the pathogenic yeast cell type, expression data for hyphal growth was compared to yeast growth in vitro and during intracellular macrophage infection. Key nutritional and cell protective pathways that show common upregulation during the yeast growth phase were identified and these included carbon and nitrogen utilization, micronutrient uptake, melanin generation and oxidative stress protection. Additionally, to separate host specific responses from temperature driven expression and identify genes that are specifically regulated during infection, macrophage infection specific transcriptional data sets were compared against the expressionprofile of T. marneffei grown at 37 ̊C in vitro. Twelve genes were chosen for phenotypic characterization using gene deletion as a way of validating the output of the screen, and these genes were shown to be important for different aspects of establishment and maintenance of T. marneffei yeast growth in macrophages. The analysis was extended for a novel gene designated msgA, encoding a guanyl nucleotide exchange factor,which was found to be essential for maintaining appropriate cell morphology, which in turn is crucial for T. marneffei occupying its macrophage niche. Overall the findings of this study revealed that T. marneffei yeast and hyphal forms have adapted specific metabolic programs tailored to the diverse environmental conditions encountered by each cell type. The identification of genes which are specifically required for establishing yeast growth during macrophage infection, uncovered components of pathways that respond to host–specific rather than temperature specific signals. Together these provide valuable insights into the initiation of infection and pathogenicity establishment in T. marneffei, and may serve to broaden our understanding of the means by which to target opportunistic fungal infections.