IDENTIFICATION OF NATURAL GENOMIC VARIANTS THAT INFLUENCE CRYPTOCOCCAL VIRULENCE

新生隐球菌是一种全球性的病原体，每年造成数十万HIV+个体死亡，并增加非艾滋病患者群体的发病率。在这种微生物的天然菌株之间观察到明显的毒力差异，但尚未报道造成这种不同毒力的天然基因组变异。这项研究的目的是填补这一知识空白。要做到这一点，需要克服当前的挑战，如单个菌株收集中菌株多样性不足，依赖多位点序列分型（MLST）来表征基因组，以及在宿主基因组水平上混淆宿主因素和潜在的发病率。我们假设，通过在标准化小鼠模型中评估一组不同的全基因组测序菌株的毒力，我们将能够通过计算识别和实验验证影响毒力的自然变异。在这个R21应用程序中，我们建议用一组初始的临床分离物来检验这一假设。两个实验室在计算生物学和实验生物学方面具有互补的技能，并且在新生芽胞杆菌方面有着富有成效的合作历史，这两个实验室的协同努力将使实现我们目标所需的分析范围成为可能。在Aim 1中，我们将从不同的临床分离株和新生C.遗传杂交后代中收集全基因组序列（WGS）和相应的小鼠感染数据。在目标2中，我们将进行全基因组关联研究（GWAS）和散装分离分析（BSA），利用我们在隐球菌生物学和基因调控方面的专业知识进行解释，以产生和优先考虑关于哪些基因组变异影响毒力的假设。在Aim 3中，我们将通过基因组工程和毒力研究直接测试高优先级假设的子集。这一应用的意义在于病原对人体健康的影响；对隐球菌基因调控和蛋白质功能的基础生物学可能的新见解；并且在疾病监测、患者分层和确定新的治疗靶点方面具有潜在的应用前景。创新方面包括使用不同的临床分离物，整体分离分析，新的计算管道，以及假设的因果变异的实验验证。总之，这些研究将为研究新生芽孢杆菌的自然变异及其在毒力中的作用提供模板，为研究界提供重要资源，并可能识别和验证影响毒力的因果变异。这一探索性建议将为我们和其他人在基础生物学理解和潜在应用方向上对更大菌株集的研究和后续工作奠定基础。

Identification of natural genomic variants that determine 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. Striking differences in virulence are observed among naturally occurring strains of this microbe, but no natural genomic variant responsible for this differential virulence has yet been reported. The goal of this research is to fill this gap in knowledge. Doing this will require overcoming the current challenges of insufficient strain diversity in individual strain collections, reli- ance on multi-locus sequence typing (MLST) to characterize genomes, and confounding host factors at the levels of the host genome and underlying morbidities. We hypothesize that by assessing the virulence of a diverse group of whole genome sequenced strains in a standardized mouse model, we will be able to computationally identify and experimentally validate natural variants that influence virulence. In this R21 application we propose to test this hypothesis with an initial set of clinical isolates. The range of analyses required to achieve our goal will be enabled by the synergistic efforts of two labs with complementary skills sets in computational and experimental biology and a history of productive collaboration on C. neoformans. In Aim 1 we will assemble whole genome sequences (WGS) and corresponding mouse infection data from diverse clinical isolates and progeny of C. neoformans genetic crosses. In Aim 2 we will perform genome-wide association studies (GWAS) and bulk segregant analysis (BSA), interpreted using our expertise in cryptococcal biology and gene regulation, to generate and prioritize hypotheses about which genomic variants influence virulence. In Aim 3 we will directly test a subset of high-priority hypotheses by genome engineering and virulence studies. The significance of this application lies in the impact of the pathogen on human health; the likely new insights into basic biology of cryptococcal gene regulation and protein function; and the potential for future application in terms of disease surveillance, patient stratification, and identification of new therapeutic targets. Innovative aspects include the use of diverse clinical isolates, bulk segregant analysis, a novel computational pipeline, and experimental validation of hypothesized causal variants. Together, these studies will provide a template for investigations of natural variants in C. neoformans and their role in virulence, generate significant resources for the research community, and potentially identify and validate causal variants that influence virulence. This exploratory proposal will additionally lay the groundwork for future studies of larger strain sets and follow-up by us and others in the directions of both fundamental biological understanding and potential application.