Early in vivo expressed antigens and their role in virulence, immune response, and vaccines for coccidioidomycosis

项目摘要/摘要(研究项目1) 新出现的和地方性霉菌病在全球范围内对人类健康的影响越来越大，人们对此知之甚少 关于大多数新出现的真菌病原体的毒力机制。球孢子虫和波萨达斯球虫 引起球孢子菌病(又名谷热)，这是特别令人担忧的，因为最近在美国迅速增加 疾病发病率在过去20年中增加了10倍以上。遣散中心- 疾病控制和预防中心(CDC)的报告称，治疗山谷热的平均成本为每个患者5万美元， 包括长时间的住院和治疗方案。它们的高级基因组和遗传分析 真菌病原体可以帮助我们提高对致病机制的理解，支持对病原菌的鉴定和鉴定。 新的生物标记物靶点的建立，并代表了开发新的生物标志物所必需的基础框架 诊断工具、治疗策略和通过接种疫苗预防疾病。目前，很少有特定的病毒- 球孢子虫的LENSE机制已被研究。使这些调查复杂化的是缺乏 活体数据，因为体外培养的生物学和基因表达分析不能复制感染性硒- 奎斯。我们已经通过对两种球虫在小鼠中的详细转录分析来弥补这一缺口 感染五天后的肺部。基于我们的转录分析和现有的基因组数据，加上硅胶 功能预测，我们已经确定了26个假定的抗原/毒力因子。这些球藻属(Coccidioidesspp.)交易- SCRIPTS在体内条件下高度表达，被预测为小分泌蛋白。我们的 工作假设是，这些蛋白质将是抗原性和潜在的毒力因子。我们将测试这一点- 构建CRISPR-Cas9基因缺失菌株。这些菌株将在两个疫区进行毒力测试。 与动物核心组织合作的MAL模型。我们将进一步研究这些基因在两者中的功能 球孢子虫的种类，特别是对寄生结构(球体)发育的影响，毒力， 以及利用纳米串在体内对宿主和病原体的反应。基因缺失菌株的RNAseq分析比较 亲本菌株将被用于比较转录网络。对这些数据的分析将提供新的信息- 洞察基因功能，并有效地将为这条路径研究的基因缺失菌株的数量增加一倍- Ogen，这将增加我们对真菌发病机制的理解。这些数据将由 识别T细胞表位和同源T细胞受体的另外两个研究项目 宿主免疫反应和可能的高级诊断测试，以及识别核的靶标 酸性疫苗。对早期感染毒力因子的免疫反应进行编程具有很大的潜力- TAL为谷热病提供保护性免疫力。总而言之，这项提议代表了一对独特的广告- 对一种重要但未被充分研究的真菌病原体进行先进的基因组数据分析和功能评估。

Project Summary/Abstract (Research Project 1) Emerging and endemic mycoses have a growing impact on human health on a global scale and little is known about virulence mechanisms for most emerging fungal pathogens. Coccidioides immitis and C. posadasii cause coccidioidomycosis (aka Valley Fever), which is of specific concern due to recent rapid increases in US disease incidence with a more than a 10-fold increase of reports over the last 20 years. The Centers for Dis- ease Control and Prevention (CDC) report that the average cost for treating Valley fever is $50,000 per patient, including extensive hospital stays and treatment regimes. Advanced genomic and genetic analyses of these fungal pathogens can help improve our understanding of mechanisms of pathogenicity, support the identifica- tion of new biomarker targets, and represent the foundational framework that is necessary for development of diagnostic tools, treatment strategies and prevention of disease via vaccination. Currently, few specific viru- lence mechanisms have been investigated in Coccidioides. Complicating these investigations is the lack of in vivo data, as the biology and gene expression analysis of in vitro cultures does not replicate the infective se- quence. We have remedied this gap by detailed transcriptional analysis of both Coccidioides species in mouse lungs five days after infection. Based on our transcriptional analyses and existing genomic data, plus in silico functional predictions, we have identified 26 putative antigens/virulence factors. These Coccidioides spp. tran- scripts are highly expressed under in vivo conditions and are predicted to be small secreted proteins. Our working hypothesis is that these proteins will be antigenic and potentially virulence factors. We will test this hy- pothesis by creating CRISPR-Cas9 gene deletion strains. These strains will be tested for virulence in two ani- mal models in collaboration with the Animal Core. We will further investigate these genes’ functions in both Coccidioides species, specifically the effects on development of the parasitic structure (spherule), virulence, and host and pathogen response in vivo using nanoString. RNAseq analysis of gene deletion strains compared to parent strain will be used to compare transcriptional networks. Analysis of these data will provide new in- sights into gene functions, and effectively double the number of gene deletion strains investigated for this path- ogen, which will increase our understanding of fungal pathogenesis mechanisms. These data will be used by the other two Research Projects to identify T cell epitopes and the cognate T cell receptors for understanding the host immune response and possible advanced diagnostic tests, as well as to identify targets for nucleic acid vaccines. Programming the immune response toward early infection virulence factors has the great poten- tial to provide protective immunity to Valley Fever. In total, this proposal represents a unique pairing of ad- vanced genomic data analysis and functional assessments in an important, yet understudied, fungal pathogen.