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.

摘要 马尔尼菲篮状菌（Talaromycesmarneffei，Tm）是引起人类疾病的七种二型病原真菌之一 在免疫功能正常和免疫功能低下的患者中， 患者Tm在整个东南亚地区流行，该地区包括超过一半的 艾滋病毒/艾滋病是全球人口中最常见的疾病，也是艾滋病毒/艾滋病患者感染和死亡的常见原因。所述生物体是 存在于环境中，并与竹鼠物种有关， 反复暴露。治疗选择有限且不理想，诊断方式仍有待改进。 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 适于通过农杆菌介导的同源重组进行转化和基因缺失 跨界（AMT）接合，很少有研究已经做了，以阐明在Tm的毒力因子。最近的一 研究报告了在一些临床分离物中存在新的dsRNA真菌病毒，并且该病毒的存在 与动物模型中增强的毒力和基因表达的下调有关 参与RNAi，一个可以靶向和降解dsRNA的过程。 在我们的建议中，我们试图利用这些进展来剖析真菌病毒和遗传学。 Tm毒力的决定因素。在目标1中，我们将分析最近发现的 在大量经充分验证的Tm人类临床分离株（N=293）中， 临床结果数据和全基因组数据可用，并评估存在的相关性 与疾病严重程度和患者预后的关系。我们将开发治疗方法 并重新引入该真菌病毒，以构建含有和不含该病毒的同类菌株， 毒力实验室分析。目的二，我们将确定真菌病毒引起的RNAi的抑制是否 在病毒感染的菌株中导致超变表型。使用转座子陷阱分析，我们将筛选 TM患者样本采集，以获得产生大量耐药菌落的分离株，并确定 如果抗药性是由于转座因子插入到负责抗药性的基因中。我们 然后将评估超变基因表型与病毒感染和未感染Tm的任何相关性 患者样本。我们将使用AMT来删除RNAi途径的组分，我们将确定 这些突变对超突变、病毒载量以及毒力是否改变的后果。这些研究将 推进我们对Tm真菌病毒和遗传毒力决定因素的理解， 对患者预后和治疗的可能影响。