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: 10619216
• 负责人: Lukasz Kozubowski
• 金额: $ 35.69万
• 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/10619216
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Address; Alleles; Animal Model; 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; Exhibits; Exposure to; Fatal Outcome; Filament; Fluconazole; Fluorescence Microscopy; Foundations; Genes; Genetic; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; HIV; High temperature of physical object; Homeostasis; Human; Hypersensitivity; Immunocompromised Host; In Vitro; Infection; Inhalation; Inositol Phosphates; Knowledge; Life; Light; Link; Lipid Bilayers; Lipids; Lung; Maintenance; Mediator of activation protein; Membrane; Membrane Fluidity; Membrane Lipids; Meningoencephalitis; Modeling; Monomeric GTP-Binding Proteins; Mus; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Population; 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; base; biophysical properties; experimental study; fluidity; genetic approach; improved; inhibitor; innovation; lipid metabolism; mortality; mutant; novel; pathogenic fungus; predictive modeling; prevent; protein complex; reconstitution; recruit; response; side effect; temperature sensitive mutant; thermozymocidin; treatment strategy

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.

项目摘要 该提案的长期目标是揭示新型隐球菌的致病机制 (Cn)专注于Cn如何适应宿主温度。Cn是一种真菌病原体，一旦进入肺部， 通过血液传播，主要在艾滋病毒/艾滋病中引起威胁生命的脑膜脑炎 患者目前的抗隐球菌疗法可能具有毁灭性的副作用。因此，新的治疗策略 确保消除隐球菌病相关的死亡率。这项建议的目的是 确定温度如何被Cn感知并传递到下游效应器通路，这些通路对于 致病性核心假设是，称为septins的形成酶的GTP酶提供了一种必需的 将温度传感与补偿信号应答和血浆连接的中枢蛋白复合物 膜（PM）稳态。有人提出，暴露于宿主温度会导致流动性增加， PM内曲率和PM相关磷脂酰肌醇4，5-二磷酸（PIP 2）升高 导致基于PIP 2结合在PM处富集septins，其在Cn适应于 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， 蛋白激酶C（PKC）途径。为了支持这一假设，使用重组septins或 采用模式生物的研究表明，隔膜通过它们的倾向识别膜曲率， 绑定PIP 2。重要的是，重组septins防止温度诱导的脂质双层组成的变化。 支持该应用的初步数据是：1）Cn PIP 2水平在37°C下增加。2)隔膜与 当Cn变为37°C时的PM。3)隔膜蛋白缺陷突变体表现出增加的PM渗透性， 对干扰PM的药物敏感。已发表的和初步的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。中心假设将通过以下方式进行检验： 追求两个目标。目的1：将使用TIRF显微镜确定PM时Septin组装的动力学。 遗传学和药理学方法将确定是否septin复合物在PM富集 基于PIP 2水平的增加Cdc 42的一个新的光开关等位基因（参与septin的GT3） 组装）将有助于建立Cdc 42下游的效应物，其在septin复合物形成中起作用。胞裂蛋白 缺乏两亲性螺旋（AH）的突变体将用于测试AH在募集septin复合物以 首相目的2：Septin复合物对PM脂质组成、PM生物物理性质、应激的影响 将确定依赖于PIP 2的应答信号传导和动物感染模型中的发病机理。的 拟议的研究是重要的，因为它将阐明一种新的机制，通过这种机制，septins有助于 感知高温和调节PM依赖的应激反应信号通路， 发病机制Cn.结果将是更好地了解Cn如何适应宿主温度， 在此基础上开发改进的抗隐球菌疗法在艾滋病毒/艾滋病患者。