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.

抽象的 隐球菌性脑膜脑炎占艾滋病患者死亡总数的 15%。 目前还没有针对隐球菌病的疫苗，这种疾病每年夺去数十万人的生命， 尽管目前的抗真菌疗法面临着预防和治疗的挑战，但全球死亡率仍高达 70%。 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。 新型隐球菌是一种环境担子菌酵母，具有在环境中生存和扩增的能力 与人类宿主生理相关的条件是其发病的先决条件。 人类的温度 (≥37°C) 和二氧化碳水平 (≥5%) 与真菌的原生环境存在显着差异 环境生态位（环境空气中的二氧化碳含量约为 0.04%）。 申请人发现，对CO2敏感的隐球菌病小鼠模型中的毒力显着减弱。 耐 CO2 临床分离株中 RAM 通路任何保守成分的破坏 H99，包括效应激酶 Cbk1，使该菌株无法在宿主温度或二氧化碳水平下生长。 一致地，cbk1突变体在鼻内感染模型和静脉内感染模型中均无毒力 因此，了解 RAM 通路如何调节隐球菌耐热性。 二氧化碳耐受性将告诉我们这种真菌如何适应宿主条件从而导致人类疾病。 由于对担子菌中 RAM 途径的下游效应子一无所知， 申请人对隐球菌 cbk1 突变体进行了初步抑制子筛选，分析了抑制子。 突变体显示，两个 RNA 加工调节因子 Ssd1 和 Psc1 中的任何一个的破坏，部分地 恢复了 cbk1 的耐热性和 CO2 耐受性，Ssd1 在真菌中是保守的，并且是已知的靶标。 Cbk1 基于对子囊菌的研究，包括模型酵母或致病性念珠菌物种。 Cbk1 缺乏磷酸化，酵母菌 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，Psc1 是一种未表征的蛋白质。 具有 PARN RNA 识别基序 PARN 基序存在于许多真核细胞谱系中，包括 担子菌真菌和人类，但令人惊讶的是在子囊菌如酵母菌或念珠菌中不存在 基于这些观察，申请人将耐热性和二氧化碳耐受性归结为物种。 隐球菌在转录后水平上至少部分受到 RAM 通路的调节。 探索性 R21 申请，申请人寻求 (1) 定义隐球菌中的 Cbk1 激酶下游靶标 通过比较磷酸化蛋白质组学和广泛的抑制子筛选，以及 (2) 识别生物过程 RAM 通路通过定义 Ssd1 和 Psc1 存在时结合的蛋白质和 mRNA 来控制 这些发现将加深我们对新型隐球菌如何适应宿主的理解。 条件，并可能揭示可用于对抗这种致命疾病的新靶标的候选物。