Investigation of key proteases in the parasitic phase of Coccidioides

球孢子菌寄生期关键蛋白酶的研究

• 批准号: 10537230
• 负责人: Anita Sil
• 金额: $ 24.23万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-02 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10537230
• 关键词: Antifungal Agents; Area; Arthrodermataceae; Aspergillus; Biological Assay; Biology; CRISPR/Cas technology; Candida; Cells; Central America; Coccidioides; Coccidioidomycosis; Communicable Diseases; Cryptococcus; Data; Data Set; Development; Developmental Biology; Developmental Process; Disease; Disease Progression; Family; Fusarium; Future; Gene Deletion; Gene Expression Profiling; Gene Family; Genes; Genetic; Goals; HIV; Hospitalization; Immunocompetent; Incidence; Individual; Infection; Inhalation; Investigation; Kinetics; Lead; Link; Metalloproteases; Methods; Molecular; Morbidity - disease rate; Morphology; Peptide Hydrolases; Phase; Phenanthrolines; Phenotype; Play; Process; Protease Inhibitor; Proteins; Publications; Reproduction spores; Research; Role; Rupture; Serine Protease; Serine Proteinase Inhibitors; Site; Soil; South America; Testing; Therapeutic; United States; Virulence; Virulence Factors; Work; Zinc; base; experience; experimental study; fungus; genetic testing; inhibitor; interest; mortality; mutant; novel therapeutics; pathogenic fungus; stem; therapeutic candidate; therapeutic target; transcriptome; transcriptome sequencing; transcriptomics

Project Summary Coccidioides spp. are major fungal pathogens endemic to the Southwest United States, Central America, and South America. In recent years, the incidence of coccidioidomycosis has continued to rise. Coccidioides infects and kills immunocompetent individuals when they inhale spores from the soil and, despite current antifungal treatment, continues to cause unacceptably high morbidity and mortality. The ability of Coccidioides to cause disease depends on an elaborate developmental transition from mycelia found in the soil to the host form known as a spherule, a morphology unique to Coccidioides. While this developmental process can be recapitulated in the lab, the molecular determinants of spherulation remain poorly understood. Delineating which genes are required for spherulation will lead to identification of therapeutic targets and accelerate discovery of new antifungals for the treatment of coccidioidomycosis. We will focus this project on two families of secreted proteases, subtilases and deuterolysins, and assess the role they play in spherulation. These protease families have undergone evolutionary expansion in the Coccidioides lineage and, since spherules are unique to Coccidioides, we hypothesize that their evolutionary expansion implicates them in the process of spherule development. Protease inhibitors have proven to be successful therapeutics for other infectious diseases, including HIV. The Coccidioides proteases that we discover to be required for the process of spherulation will be excellent candidates for therapeutic targets. We will use a multipronged approach, including transcriptional profiling, protease inhibitor studies, and genetics, to elucidate the role of key proteases in spherulation. This will leverage PI Sil’s extensive experience with transcriptional profiling of dimorphic fungi, including Coccidioides, and collaborator Dr. Craik’s expertise on the biology of proteases, including previous work on secreted proteases in pathogenic fungi. In Aim 1, we will identify candidate secreted proteases involved in spherulation by profiling the transcriptome of multiple developmental stages of Coccidioides to identify proteases whose expression is spherule-enriched. Additionally, we will determine the effect of class-wide protease inhibitors on spherule formation, thereby implicating individual protease families as key players in Coccidioides spherule development. In Aim 2, we will generate six mutant Coccidioides strains, each lacking a secreted protease which is already implicated in spherulation from our preliminary studies. Additionally, we will generate six more protease mutants based on prioritization from studies in Aim 1. Using these deletion mutants, we will test whether each protease is required for spherulation. Our work will provide a rich transcriptional profiling dataset as well as multiple deletion mutants that are critical for dissecting the role of these two expanded protease families in the parasitic phase of Coccidioides biology.

项目摘要 球藻(Coccidioidesspp.)是美国西南部、中美洲和 南美。近年来，球孢子菌病的发病率持续上升。球孢子虫感染 当免疫能力强的人从土壤中吸入孢子时会杀死他们，尽管目前有抗真菌药物 治疗方面，继续造成令人无法接受的高发病率和死亡率。球虫的致病能力 疾病依赖于从土壤中发现的菌丝体到已知的寄主形式的复杂的发育转变 球状，球状球虫特有的一种形态。虽然这一发展过程可以概括为 在实验室，球化的分子决定因素仍然知之甚少。描述哪些基因是 所需的球化将导致识别治疗靶点并加速发现新的 治疗球孢子菌病的抗真菌药物。我们将把这个项目集中在两个秘而不宣的家庭 蛋白水解酶、枯草杆菌酶和二氢解氨酶，并评估它们在球化中所起的作用。这些蛋白酶家族 在球形目的谱系中经历了进化的扩展，由于球体是 球虫，我们假设它们的进化扩张牵涉到球体的形成过程 发展。蛋白酶抑制剂已被证明是治疗其他感染性疾病的成功药物， 包括艾滋病毒。我们发现球状芽孢杆菌在球化过程中所需的蛋白水解酶 治疗靶点的极佳候选者。 我们将使用多管齐下的方法，包括转录图谱、蛋白水解酶抑制剂研究，以及 遗传学，阐明关键的蛋白水解酶在球化中的作用。这将利用皮西尔的丰富经验 包括球孢子菌在内的二相性真菌的转录图谱，以及合作者Craik博士在 蛋白酶的生物学，包括以前关于病原真菌中分泌的蛋白酶的工作。在目标1中，我们将 通过对多个转录组的分析确定参与球化的候选分泌型蛋白水解酶 球孢子虫的发育阶段，以确定其表达是球状丰富的蛋白水解酶。另外， 我们将确定全类蛋白水解酶抑制剂对球体形成的影响，从而影响个体 蛋白水解酶家族在球状球虫球体发育中的关键作用。在目标2中，我们将产生6个突变体 球孢子虫的每个菌株都缺乏一种分泌的蛋白酶，这种酶已经与我们的球状结构有关 初步研究。此外，我们将根据研究的优先顺序再产生六个蛋白酶突变体 在目标1中，使用这些缺失突变体，我们将测试是否每个蛋白酶都是球化所必需的。我们的工作 将提供丰富的转录图谱数据集以及多个删除突变体，这对 剖析这两个扩展的蛋白水解酶家族在球孢子虫生物学寄生阶段中的作用。