Implications of mycoviral infection in Talaromyces marneffei: an analysis of human patient samples, RNAi, and hypermutation

马尔尼菲踝节菌中真菌病毒感染的影响：对人类患者样本、RNAi 和超突变的分析

• 批准号: 10191218
• 负责人: JOSEPH HEITMAN
• 金额: $ 20.13万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-02 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10191218
• 关键词: AIDS/HIV problem; Agrobacterium; Alleles; Animal Model; Automobile Driving; Biological; Biological Assay; Biology; Blood; Cessation of life; Clinical; Collection; Congenic Strain; DNA Transposable Elements; Data; Development; Diagnostic; Disease Management; Double Stranded RNA Virus; Double-Stranded RNA; Down-Regulation; Drug resistance; Environment; Exposure to; FK506; Flucytosine; Foundations; Gene Deletion; Gene Expression Regulation; Genes; Genetic; Genetic Determinism; Genetic study; Genome; Genomic approach; Geographic Locations; Goals; Growth; Health; Human; Immunocompetent; Immunocompromised Host; Incubated; Infection; Insertional Mutagenesis; Investigation; Laboratories; Life; Link; Malassezia; Mediating; Methods; Microbiology; Modality; Modeling; Modification; Molecular Genetics; Morbidity - disease rate; Movement; Mus; Mutation; Mycoses; Neomycin; Nucleic Acids; Organism; Outcome; Parasites; Pathogenesis; Pathogenicity; Pathologic Mutagenesis; Pathway interactions; Patient Isolators; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Phenotype; Plasmids; Play; Population; Process; Prognosis; Publishing; RNA Interference; Rattus; Recurrent disease; Reporting; Research; Resistance; Risk; Role; Sampling; Severity of illness; Sirolimus; Southeastern Asia; Talaromyces; Temperature; Testing; The science of Mycology; Therapeutic; Untranslated RNA; Viral; Viral Load result; Virulence; Virulence Factors; Virus; Work; antimicrobial drug; base; clinical diagnostics; disorder control; drug isolation; experience; experimental study; follow-up; fungus; gel electrophoresis; genetic approach; genome sequencing; homologous recombination; human disease; hygromycin A; improved; insight; mortality; mouse model; mutant; novel; particle; pathogenic fungus; pathogenic microbe; prognostic; prospective; relapse patients; sample collection; screening; treatment response; whole genome

Abstract Talaromyces marneffei (Tm) is one of seven dimorphic human fungal pathogens that are causative agents of substantial global morbidity and mortality in both immunocompetent and immunocompromised patients. Tm is endemic throughout Southeast Asia in a geographic region that encompasses more than half of the global population, and is a frequent cause of infection and death in HIV/AIDS patients. The organism is present in the environment and in association with bamboo rat species and there is substantial risk of frequent and repeated exposure. Treatment options are limited and suboptimal, diagnostic modalities remain to be optimized, and our understanding of virulence determinants is limited. Although studies have shown that Tm is amenable to transformation and gene deletion via homologous recombination through Agrobacterium mediated trans-kingdom (AMT) conjugation, little investigation has been done to elucidate virulence factors in Tm. A recent study reported the presence of a novel dsRNA mycovirus in some clinical isolates, and that presence of this virus is correlated with enhanced virulence in animal models and the down regulation of the expression of genes involved in RNAi, a process which can target and degrade dsRNAs. In our proposal, we seek to capitalize upon these advances in order to dissect the mycoviral and genetic determinants of Tm virulence. In aim 1, we will analyze the presence or absence of the recently discovered mycovirus in a large collection (N=293) of well-validated Tm human clinical isolates with robust mycological and clinical outcome data and for which whole-genome data is available, and to assess correlations of the presence of this mycovirus determinant with disease severity and patient outcomes. We will develop approaches to cure and reintroduce this mycovirus in order to construct congenic strains with and without the virus for direct laboratory analyses of virulence. In aim 2, we will determine if the supression of RNAi caused by the mycovirus results in a hypermutator phenotype in virus-infected strains. Using a transposon trap assay, we will screen the Tm patient sample collection for isolates that produce a larger number of drug-resistant colonies, and determine if drug resistance is due to the insertion of transposable elements into the gene responsible for resistance. We will then assess any correlations of the hypermutator phenotype with virus-infected and virus-uninfected Tm patient samples. We will use AMT to delete components of the RNAi pathway and we will determine the consequences of these mutations on hypermutation, viral load, and if virulence is altered. These studies will advance our understanding of Tm mycoviral and genetic virulence determinants and provide insights with possible implications for patient prognosis and treatment.

抽象的 Talaromyces Marneffei（TM）是七个是二态人类真菌病原体之一，它们是病原体 免疫能力和免疫功能低下的全球发病率和死亡率的药物 患者。 TM在整个东南亚都是地方性的地理区域，该地区包括超过一半的地理区域 全球人口，是艾滋病毒/艾滋病患者感染和死亡的经常原因。有机体是 存在于环境中并与竹大鼠物种相关，并且存在频繁的很大风险 并反复接触。治疗方案有限，次优，诊断方式仍然是 优化，我们对毒力决定因素的理解是有限的。尽管研究表明TM是 通过农杆菌介导的同源重组来适应转化和基因缺失 跨王朝（AMT）共轭，几乎没有研究以阐明TM中的毒力因子。最近 研究报道了在某些临床分离株中存在新型DSRNA菌病毒的存在，该病毒的存在 与动物模型中的毒力增强和基因表达的下调相关 参与RNAi，该过程可以靶向和降解DSRNA。 在我们的提案中，我们试图利用这些进步，以剖析霉菌病毒和遗传 TM毒力的决定因素。在AIM 1中，我们将分析最近发现的存在或不存在 在大量验证的TM人类临床分离株中，有牢固的真菌学和稳健的真菌学和 临床结果数据以及可获得的全基因组数据，并评估存在的相关性 这是疾病严重程度和患者结局的霉菌病毒决定因素。我们将开发治愈方法 并重新引入该菌病毒，以便在有或没有病毒的情况下构建过同基菌株 毒力的实验室分析。在AIM 2中，我们将确定RNAi是否由Mycovirus引起 导致病毒感染菌株中的高压表型。使用转座陷阱测定法，我们将筛选 TM患者样品收集用于产生较大药物抗性菌落的分离株，并确定 如果耐药性是由于将转座元素插入负责抗性的基因中。我们 然后，将评估高压表型的任何相关性与病毒感染的病毒未感染的TM 病人样本。我们将使用AMT删除RNAi途径的组件，我们将确定 这些突变对超突变，病毒载量和毒力改变的后果。这些研究会 促进我们对TM霉菌病毒和遗传毒力决定因素的理解，并提供见解 对患者预后和治疗的可能影响。