Natural genomic variants that influence cryptococcal pathogenicity

项目摘要 影响隐球菌致病性的天然基因组变异 新型隐球菌是一种全球性病原体，每年造成数十万人死亡， 在非艾滋病患者人群中，艾滋病的发病率不断上升。C.从宾夕法尼亚州收集的新形虫菌株， 隐球菌脑膜炎的病例已被用来阐明C.新形式的进化和努力，以纠正- 晚期疾病的结果与体外措施，如毒力因子的生产或真菌生长。我们知道 不同的基因组序列与不同的临床结果相关。然而，我们不知道哪种自然 基因组变异是临床分离株的不同毒力的原因。 我们的长期目标是了解隐球菌属细菌的自然变异的分子机制， 基因组决定致病性。实现这一目标的第一步是识别自然发生的变异， 在单基因分辨率下，实验证明会影响毒力。下一步是表征 这些基因变异对隐球菌生物学和疾病进展的影响。我们假设我们可以 联合收割机遗传和基因组方法与实验跟踪相结合，以识别，验证和表征天然 聚集发生的变异显著影响隐球菌毒力。这一假设得到了我们的支持。 令人信服的初步数据，其中两种不同的遗传方法涉及同一基因组区域的变异 影响传染性。 我们建议通过追求三个目标来实现我们的目标：目标1是使用两个不同的，互补的统计 发现含有影响真菌维鲁的天然变异体的基因组区域的遗传学方法， lence。目标2是进一步应用遗传学，通过表达分析和突变体研究， 区域，并确定其中特定的高优先级基因。目的3是利用基因组工程来验证 选择的变异基因对毒力的影响，并评估其对真菌基因表达，生理， ogy和小鼠模型中的致病性。 应用公正的策略来识别携带影响毒力的自然变异的基因， 抗真菌治疗的新的潜在靶点。对这些变体进行表征也将为未来的投资铺平道路。 它们的作用的机制基础，这将阐明隐球菌生物学的关键特征， 发病机制我们的方法适用于隐球菌生物学中的其他基本问题，并可能 特别是其他微生物病原体。最后，我们的研究将产生有价值的试剂和数据集， 加强其他C. neoformans研究人员。

PROJECT SUMMARY Natural genomic variants that influence cryptococcal pathogenicity Cryptococcus neoformans is a global pathogen responsible for hundreds of thousands of deaths yearly in HIV+ individuals and increasing morbidity in non-AIDS patient populations. C. neoformans strains collected from pa- tients with cryptococcal meningitis have been used to elucidate C. neoformans evolution and in efforts to corre- late disease outcome with in vitro measures such as virulence factor production or fungal growth. We know that distinct genome sequences are associated with varied clinical outcomes. However, we do not know which natural genomic variants are responsible for the differential virulence of clinical isolates. Our long-term goal is to understand the molecular mechanisms by which natural variation in the cryptococcal genome determines pathogenicity. A first step toward this goal is to identify naturally occurring variants that can be experimentally proven to influence virulence, at single-gene resolution. The next step is to characterize the impact of variants in these genes on cryptococcal biology and disease progression. We hypothesize that we can combine genetic and genomic methods with experimental follow-up to identify, validate, and characterize natu- rally occurring variants that significantly influence cryptococcal virulence. This hypothesis is supported by our compelling preliminary data, in which two distinct genetic methods implicate variants in the same genomic region as influencing infectivity. We propose to achieve our goals by pursuing three Aims: Aim 1 is to use two distinct, complementary statistical genetics methods to discover genomic regions containing naturally occurring variants that influence fungal viru- lence. Aim 2 is to further apply genetics, supported by expression analysis and mutant studies, to refine these regions and identify specific high-priority genes within them. Aim 3 is to use genome engineering to validate the influence of selected variant genes on virulence and to assess their effects on fungal gene expression, physiol- ogy, and pathogenicity in a mouse model. Applying an unbiased strategy to identify genes harboring natural variants that influence virulence will highlight new potential targets for antifungal therapy. Characterizing these variants will also pave the way for future inves- tigations of the mechanistic basis of their effects, which will elucidate key features of cryptococcal biology and pathogenesis. Our methods are applicable to other fundamental questions in cryptococcal biology, and poten- tially to other microbial pathogens. Finally, our studies will produce valuable reagents and data sets that will enhance investigations by other C. neoformans researchers.