Define the molecular bases for cryptococcal adaptation to host conditions by the RAM pathway

通过 RAM 途径定义隐球菌适应宿主条件的分子基础

• 批准号: 10627371
• 负责人: Xiaorong Lin
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627371
• 关键词: ACE2; Acquired Immunodeficiency Syndrome; Air; Antifungal Agents; Antifungal Therapy; Ascomycota; Basidiomycota; Binding; Binding Proteins; Biological Process; Candida; Carbon Dioxide; Cells; Central Nervous System Diseases; Cessation of life; Chemicals; Clinical; Complex; Critical Pathways; Cryptococcosis; Cryptococcus; Cryptococcus neoformans; Cultural Backgrounds; Development; Disease; Exclusion; Future; Genes; Goals; Growth; High temperature of physical object; Homologous Gene; Human; Immunoprecipitation; Infection; Intravenous; Investigation; Meningoencephalitis; Messenger RNA; Microbe; Modeling; Molecular; Morphogenesis; Mutagens; Names; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Pilot Projects; Post-Transcriptional Regulation; Process; Proteins; Public Health; RNA; RNA Processing; RNA Recognition Motif; Research; Saccharomyces; Suppressor Mutations; Temperature; Transcript; Translational Repression; Translations; Virulence; Work; Yeast Model System; Yeasts; attenuation; base; comparative; fungus; genetic analysis; improved; mRNA Translation; mortality; mouse model; mutant; novel; novel therapeutics; pathogenic fungus; phosphoproteomics; posttranscriptional; prevent; response; stress granule; trait; transcription factor; transcriptome sequencing; vaccine access

Abstract Cryptococcal meningoencephalitis is responsible for 15% of the total deaths of AIDS patients. There is no vaccine available for cryptococcosis and the disease claims hundreds of thousands of lives each year, with the global mortality rates of ~70% despite current antifungal therapies. The challenges of preventing and treating this disease motivate the investigation of cryptococcal pathways that are critical for pathogenesis. As Cryptococcus neoformans is an environmental basidiomycetous yeast, the ability to survive and amplify in conditions physiologically relevant for the human host is a prerequisite for its pathogenesis. Both high temperatures (≥37C) and CO2 levels (≥5%) in humans differ considerably from the fungus' primary environmental niches (CO2 in ambient air is ~0.04%). Accordingly, environmental cryptococcal isolates that are CO2-sensitive showed drastic virulence attenuation in mouse models of cryptococcosis. The applicant found that disruption of any of the conserved components of the RAM pathway in the CO2-tolerant clinical isolate H99, including the effector kinase Cbk1, rendered the strain unable to grow at host temperatures or CO2 levels. Consistently, the cbk1 mutant is avirulent in both an intranasal infection model and an intravenous infection model of cryptococcosis. Thus, understanding how the RAM pathway regulates cryptococcal thermotolerance and CO2 tolerance will inform us about how this fungus adapts to host conditions to cause diseases in humans. As nothing is known about downstream effectors of the RAM pathway in basidiomycete fungi, the applicant carried out a pilot suppressor screen of the cryptococcal cbk1 mutant. Analyzing the suppressor mutants revealed that disruption of either of the two RNA-processing regulators, Ssd1 and Psc1, partially restored cbk1's thermotolerance and CO2 tolerance. Ssd1 is conserved among fungi and a known target of Cbk1 based on studies in ascomycetes, including model yeasts or pathogenic Candida species. In the absence of phosphorylation by Cbk1, Saccharomyces Ssd1 translocates to processing or P-bodies and stress granules, thereby suppressing translation of its bound mRNAs. By contrast, Psc1 is an uncharacterized protein with a PARN RNA-recognition motif. The PARN motif is present in many eukaryotic lineages including basidiomycete fungi and humans, but surprisingly absent in ascomycetes such as Saccharomyces or Candida species. Based on these observations, the applicant hypothesizes that thermotolerance and CO2 tolerance in Cryptococcus are regulated by the RAM pathway  at least partly  at the post-transcriptional level. In this exploratory R21 application, the applicant seeks to (1) define Cbk1 kinase downstream targets in Cryptococcus by comparative phosphoproteomics and extensive suppressor screens and (2) identify biological processes controlled by the RAM pathway by defining the proteins and mRNAs bound by Ssd1 and Psc1 in the presence and absence of Cbk1. These findings will deepen our understanding of how C. neoformans adapts to host conditions and may reveal candidates that can be exploited for novel targets against this deadly disease.

摘要 隐球菌脑膜脑炎占艾滋病患者死亡总数的15%。有 没有疫苗可用于隐球菌病，这种疾病每年夺去数十万人的生命， 尽管目前有抗真菌治疗，全球死亡率仍高达70%。预防和 治疗这种疾病激发了对发病机制至关重要的隐球菌途径的研究。作为 新型隐球菌是一种环境担子菌酵母菌，在环境中存活和扩增的能力 与人类宿主生理相关的条件是其发病的先决条件。较高 人体的温度（≥37 ℃）和二氧化碳水平（≥5%）与真菌的主要致病菌有很大差异。 环境生态位（环境空气中的CO2约为0.04%）。因此，环境隐球菌分离物， CO2敏感型在隐球菌病小鼠模型中表现出剧烈的毒力衰减。申请人发现 在CO2耐受性临床分离株中， H99，包括效应激酶Cbk1，使菌株无法在宿主温度或CO2水平下生长。 因此，cbk 1突变体在鼻内感染模型和静脉内感染模型中均为无毒力的 隐球菌病模型。因此，了解RAM途径如何调节隐球菌的耐热性， 和二氧化碳耐受性将告诉我们这种真菌是如何适应宿主环境从而导致人类疾病的。 由于对担子菌类真菌中RAM途径的下游效应物一无所知， 申请人进行了隐球菌CBK 1 β突变体的中试抑制筛选。分析抑制器 突变体显示，两种RNA加工调节因子Ssd 1和Psc 1中的任何一种的破坏， 恢复了cbk 1的耐热性和CO2耐受性。Ssd 1在真菌中是保守的，并且是已知的 Cbk 1基于子囊菌的研究，包括模式酵母或致病性念珠菌属。在 在Cbk 1磷酸化缺失的情况下，酵母Ssd1易位至加工或P体和应激 颗粒，从而抑制其结合的mRNA的翻译。相比之下，Psc 1是一种未表征的蛋白质， 带有PARN RNA识别基序PARN基序存在于许多真核生物谱系中，包括 但令人惊讶的是，在子囊菌纲如酵母菌属或念珠菌属中不存在 物种基于这些观察结果，申请人假设，小鼠的热耐受性和CO2耐受性与小鼠的热耐受性和CO2耐受性相关。 隐球菌受RAM途径的调节，至少部分地在转录后水平上调节。在这 作为探索性R21申请，申请人寻求（1）定义隐球菌中的Cbk 1激酶下游靶点 通过比较磷酸化蛋白质组学和广泛的抑制剂筛选和（2）鉴定生物过程 通过定义Ssd 1和Psc 1存在下结合的蛋白质和mRNA， 没有Cbk1。这些发现将加深我们对C.新变型适应宿主 条件，并可能揭示候选人，可以利用新的目标，对这种致命的疾病。