Impact of RNAi and unisexual reproduction on Cryptococcus evolution, drug resistance, and pathogenesis

摘要 新生隐球菌和加蒂隐球菌是全球分布的真菌病原体，可引起 免疫功能低下和免疫能力正常的患者中，每年有220,000例危及生命的感染，主要是 在低收入国家，18万人死亡，占所有与艾滋病毒/艾滋病相关的死亡人数的15%，死亡率为70%。在研究中 在该奖项的支持下，a-和-有性生殖的范围和功能在进化和 对隐球菌的致病机理进行了界定。有性繁殖控制着种群结构，使基因 交流和促进克隆，并导致产生具有感染性的孢子。 在这一持续的筹资期间，这些机制取得了重大进展 以及有性繁殖对这些病原体及其毒力的影响：1)发现RNAi在 有丝分裂和减数分裂过程中转座子运动和转基因沉默的控制及其特性 RNAi缺失对基因组结构和稳定性的影响；2)RNAi基因的产生和影响 非整倍体对耐药性、致病性和发育的影响以及倍体传感器的工程设计 在单性生殖过程中导致内复制的遗传因素；3)引起 太平洋西北部的加蒂亚氏菌暴发和紧密排列的变异体谱系，以及4)演示 这种单性生殖产生新的遗传多样性，并增强对营养和 交配伙伴。在目前的方案中，我们假设1)RNAi在独特的分离株中被保留和丢失 和物种，影响基因组稳定性，推动耐药性和致病机制的演变；2) 单性繁殖是隐球菌病原体进化、全球分布和影响的主要力量。 我们的研究揭示了RNAi和有性繁殖的独特方面，使我们能够提出新的测试目标 这些假设。目的1重点研究RNAi在基因组稳定性中的作用，阐明RNAi的组成和作用机制 其中RNAi控制转座子并沉默重复的DNA序列，并研究短期和长期 RNAi缺失对新生葡萄球菌临床耐药和致病机制的长期影响 分离株和RNAi缺陷的Durogattii暴发种。目标2将阐明单性生殖 涉及1)混合交配型种群单性生殖的途径和机制 在将基因组重排作为物种形成模式的菌株中，2)遗传和环境因素 促进全球A型血清谱系的单性繁殖和动物模型中感染孢子的分析， 以及3)克服希尔-罗伯逊效应以实现有益突变的链接和分离 有害和有益的突变，以增强体质和致病能力。这些研究将推动 了解基因组稳定性和单性生殖如何推动病原体进化和种群 结构、抗药性的发展、侵染性孢子的产生和毒力，具有直接意义 用于传染病的演变、治疗和预防。

Abstract Cryptococcus neoformans and Cryptococcus gattii are globally distributed fungal pathogens that cause >220,000 life-threatening infections each year in immunocompromised and immunocompetent patients, leading to >180,000 deaths, >15% of all HIV/AIDS-related deaths, and >70% mortality in low-income countries. In studies supported by this award, the scope and functions of a- and - sexual reproduction on the evolution and pathogenesis of Cryptococcus were defined. Sexual reproduction governs population structure, enabling genetic exchange and promoting clonality, and results in the production of infectious spores. During this sustained funding period, significant advances were achieved characterizing the mechanisms and impact of sexual reproduction on these pathogens and their virulence: 1) discovery of roles of RNAi in controlling transposon movement and transgene silencing during mitosis and meiosis and characterization of the consequences of RNAi loss on genome structure and stability; 2) studies on generation and affect of aneuploidy on drug resistance, pathogenicity, and development, and engineering of a ploidy sensor to analyze genetic factors contributing to endoreplication during unisexual reproduction; 3) delineation of isolates causing the C. gattii outbreak in the Pacific Northwest and closely aligned hypermutator lineages, and 4) demonstration that unisexual reproduction generates genetic diversity de novo and enhances competitiveness for nutrients and mating partners. In the current proposal, we hypothesize 1) RNAi has been retained and lost in unique isolates and species, influencing genomic stability and driving evolution of drug resistance and pathogenesis, and 2) unisexual reproduction is a major force in evolution, global distribution, and impact of Cryptococcus pathogens. Our studies reveal unique facets of RNAi and sexual reproduction enabling us to propose new aims to test these hypotheses. Aim 1 focuses on RNAi in genome stability, elucidating components and mechanisms by which RNAi controls transposons and silences repetitive DNA sequences, and studying the short-term and long- term consequences of RNAi loss on drug resistance and pathogenesis in C. neoformans hypermutator clinical isolates and the RNAi-deficient C. deuterogattii outbreak species. Aim 2 will elucidate unisexual reproduction pathways and mechanisms involving 1) unisexual reproduction occurring in mixed mating-type populations and in strains with rearranged genomes as a model of speciation, 2) genetic and environmental factors that promote unisexual reproduction of global serotype A lineages and analysis of infectious spores in animal models, and 3) overcoming the Hill-Robertson effect to enable linkage of beneficial mutations and separation of detrimental and beneficial mutations to enhance fitness and pathogenesis. These studies will advance the understanding of how genome stability and unisexual reproduction drive pathogen evolution and population structure, development of drug resistance, and infectious spore production and virulence, with direct implications for infectious disease evolution, treatment, and prevention.