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: 10633259
• 负责人: Anita Sil
• 金额: $ 20.19万
• 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/10633259
• 关键词: Acceleration; 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; candidate identification; experience; experimental study; fungus; inhibitor; interest; mortality; mutant; novel therapeutics; pathogenic fungus; stem; therapeutic candidate; therapeutic target; transcriptome sequencing; transcriptomic profiling; 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 球孢子菌属是美国西南部、中美洲和 南美洲.近年来，球孢子菌病的发病率持续上升。球孢子菌属感染 并杀死免疫功能正常的人，当他们吸入孢子从土壤中，尽管目前的抗真菌 治疗方面的不足继续造成令人无法接受的高发病率和死亡率。球孢子菌引起 疾病依赖于从土壤中发现的菌丝体到已知的宿主形式的复杂发育转变 球孢子菌属特有的一种形态虽然这一发展过程可以概括为 在实验室中，对成球的分子决定因素仍然知之甚少。描述哪些基因 成球所需的药物将导致治疗靶点的识别并加速新药物的发现 用于治疗球孢子菌病的抗真菌剂。我们将把这个项目的重点放在两个家庭的分泌 蛋白酶，枯草杆菌酶和deuterolysins，并评估它们在成球中发挥的作用。这些蛋白酶家族 在球孢子菌属谱系中经历了进化扩张，由于小球是球孢子菌属特有的， 球孢子菌属，我们推测它们的进化扩张暗示它们在小球的过程中 发展蛋白酶抑制剂已被证明是其他感染性疾病的成功治疗剂， 包括艾滋病毒。我们发现球孢子菌蛋白酶在成球过程中是必需的， 治疗靶点的优秀候选者。 我们将使用多管齐下的方法，包括转录谱分析，蛋白酶抑制剂研究， 遗传学，以阐明关键蛋白酶在成球中的作用。这将利用PI Sil的丰富经验 通过对包括球孢子菌在内的二型真菌的转录谱分析，以及合作者Craik博士在以下方面的专业知识， 蛋白酶的生物学，包括以前对病原真菌分泌蛋白酶的研究。在目标1中，我们 通过分析多个转录物的转录组来鉴定参与成球的候选分泌蛋白酶， 球孢子菌属的发育阶段，以鉴定其表达是小球富集的蛋白酶。此外，本发明还 我们将确定类蛋白酶抑制剂对小球形成的影响，从而涉及个体 蛋白酶家族是球孢子菌小球体发育的关键参与者。在目标2中，我们将产生六个突变体， 球孢子菌属菌株，每一种都缺乏一种分泌的蛋白酶，这种蛋白酶已经与我们的 初步研究。此外，我们将根据研究的优先顺序再产生六种蛋白酶突变体 目标1。使用这些缺失突变体，我们将测试每种蛋白酶是否是成球所必需的。我们的工作 将提供丰富的转录谱数据集以及多个缺失突变体，这些突变体对于 剖析了这两个扩展的蛋白酶家族在球孢子菌生物学寄生阶段的作用。