The role of septins in the adaptation of Cryptococcus neoformans to host temperature in HIV-based cryptococcosis

脓毒症在 HIV 隐球菌病中新型隐球菌适应宿主温度中的作用

• 批准号: 10708985
• 负责人: Lukasz Kozubowski
• 金额: $ 38.27万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708985
• 关键词: Acquired Immunodeficiency Syndrome; Address; Alleles; Animals; Aspergillus nidulans; Binding; Blood Circulation; Cell Membrane Permeability; Cell Wall; Cell membrane; Cells; Central Nervous System Infections; Cessation of life; Complex; Critical Pathways; Cryptococcosis; Cryptococcus; Cryptococcus neoformans; Cues; Cytokinesis; Data; Drug Targeting; Engineering; Exclusion; Exhibits; Exposure to; Fatal Outcome; Filament; Fluconazole; Fluorescence Microscopy; Foundations; Genes; Genetic; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; HIV; HIV/AIDS; High temperature of physical object; Homeostasis; Human; Immunocompromised Host; In Vitro; Infection; Inhalation; Inositol Phosphates; Knowledge; Life; Light; Link; Lipid Bilayers; Lipids; Lung; Maintenance; Mediator; Membrane; Membrane Fluidity; Membrane Lipids; Meningoencephalitis; Modeling; Monomeric GTP-Binding Proteins; Mus; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Population; Proliferating; Propidium Diiodide; Protein Kinase C; Proteins; Public Health; Publishing; Recombinants; Research; Role; Signal Pathway; Signal Transduction; Sodium Dodecyl Sulfate; Sphingolipids; Sterols; Stress; Stress Response Signaling; Temperature; Temperature Sense; Testing; Time; Virulence; biophysical properties; experimental study; fluidity; genetic approach; improved; inhibitor; innovation; lipid metabolism; model organism; mortality; mutant; novel; novel therapeutic intervention; pathogenic fungus; pharmacologic; predictive modeling; prevent; protein complex; reconstitution; recruit; response; side effect; temperature sensitive mutant; thermozymocidin; transmission process

PROJECT SUMMARY The long-term goal of this proposal is to uncover mechanisms of pathogenicity of Cryptococcus neoformans (Cn) focusing on how Cn adapts to host temperature. Cn is a fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life-threatening meningo-encephalitis primarily in HIV/AIDS patients. Current anti-cryptococcal therapies can have devastating side effects. Thus, new treatment strategies are warranted to eliminate mortality associated with cryptococcosis. The objective of this proposal is to determine how the temperature is sensed by Cn and transmitted to downstream effector pathways critical for pathogenicity. The central hypothesis is that filament-forming GTP-ases called septins provide an essential hub protein complex which links temperature sensing to a compensatory signaling response and plasma membrane (PM) homeostasis. It is proposed that exposure to host temperature results in increased fluidity and curvature within the PM and the elevation of PM-associated phosphatidylinositol 4,5-bisphosphate (PIP2) leading to enrichment of septins at the PM based on PIP2 binding, which has two-fold role in Cn adaptation to host temperature: it maintains PM homeostasis and facilitates stress signaling via phospholipase C (PLC), and protein kinase C (PKC) pathways. In support of this hypothesis, experiments with recombinant septins or studies employing model organisms suggest that septins recognize membrane curvature via their propensity to bind PIP2. Importantly, recombinant septins prevent temperature-induced changes in lipid bilayer composition. Preliminary data supporting this application are: 1) Cn PIP2 levels increase at 37°C. 2) Septins associate with the PM when Cn is shifted to 37°C. 3) Septin-deficient mutants exhibit increased PM permeability and sensitivity to drugs that perturb PM. Published and preliminary data also demonstrate phenotypic similarity between septin-deficient and PLC signaling-defective mutants. The central hypothesis will be tested by pursuing two aims. Aim1: Dynamics of septin assembly at the PM will be defined with TIRF microscopy. Genetic and pharmacological approaches will determine whether septin complexes are enriched at the PM based on increased levels of PIP2. An innovative light switchable allele of Cdc42 (GTPase involved in septin assembly) will help to establish effectors down-stream of Cdc42 acting in septin complex formation. A septin mutant lacking the amphiphatic helix (AH) will be utilized to test the role of AH in recruiting septin complex to the PM. Aim2: An impact of septin complexes on PM lipid composition, PM biophysical properties, stress response signaling dependent on PIP2, and pathogenesis in animal infection model will be determined. The proposed research is significant because it will elucidate a novel mechanism through which septins contribute to sensing high temperature and regulating PM-dependent stress response signaling pathways crucial for the pathogenesis of Cn. Outcomes will be better understanding of how Cn adapts to host temperature and a new foundation upon which to develop improved anti-cryptococcal therapies in HIV/AIDS patients.

项目概要 该提案的长期目标是揭示新型隐球菌的致病机制 (Cn) 关注 Cn 如何适应宿主温度。 Cn 是一种真菌病原体，一旦进入肺部， 通过血流传播，导致危及生命的脑膜脑炎，主要发生在艾滋病毒/艾滋病中 患者。目前的抗隐球菌疗法可能会产生毁灭性的副作用。因此，新的治疗策略 有必要消除与隐球菌病相关的死亡率。该提案的目的是 确定 Cn 如何感知温度并将其传输到下游效应器通路，这对于 致病性。核心假设是，称为脓毒蛋白（septins）的丝状形成 GTP 酶提供了一种必需的 将温度传感与补偿信号响应和血浆连接起来的中心蛋白复合物 膜（PM）稳态。有人提出，暴露于宿主温度会导致流动性增加， PM 内的曲率和 PM 相关磷脂酰肌醇 4,5-二磷酸 (PIP2) 的升高 基于 PIP2 结合，导致 PM 处脓毒蛋白的富集，这在 Cn 适应中具有双重作用 宿主温度：它维持 PM 稳态并通过磷脂酶 C (PLC) 促进应激信号传导，以及 蛋白激酶 C (PKC) 途径。为了支持这一假设，用重组脓毒症或 使用模型生物的研究表明，脓毒症通过其倾向于识别膜曲率 绑定 PIP2。重要的是，重组败血症可防止温度引起的脂质双层组成的变化。 支持该应用的初步数据为：1) Cn PIP2 水平在 37°C 时增加。 2) Septins 与 当 Cn 移动到 37°C 时的 PM。 3) Septin缺陷突变体表现出增加的PM渗透性和 对干扰 PM 的药物的敏感性。已发表的数据和初步数据也证明了表型相似性 septin 缺陷型和 PLC 信号缺陷型突变体之间的差异。中心假设将被检验 追求两个目标。目标 1：PM 处的 Septin 组装动力学将通过 TIRF 显微镜来定义。 遗传和药理学方法将确定 Septin 复合物是否在 PM 时富集 基于 PIP2 水平的增加。 Cdc42 的创新光可切换等位基因（参与 septin 的 GTPase） 组装）将有助于建立 Cdc42 下游作用于 septin 复合物形成的效应子。脓毒症 缺乏两亲螺旋 (AH) 的突变体将用于测试 AH 在招募 septin 复合物以 总理。目标 2：septin 复合物对 PM 脂质成分、PM 生物物理特性、应激的影响 依赖于 PIP2 的反应信号以及动物感染模型中的发病机制将被确定。这 拟议的研究意义重大，因为它将阐明脓毒症贡献的一种新机制 感测高温并调节 PM 依赖性应激反应信号通路，这对于 Cn 的发病机制。结果将是更好地了解 Cn 如何适应宿主温度和新的 在此基础上开发改进的艾滋病毒/艾滋病患者抗隐球菌疗法。