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.

项目总结 这项提案的长期目标是揭示新生隐球菌的致病机制。 (CN)重点研究CN如何适应寄主温度。CN是一种真菌病原体，当进入肺部和 通过血液传播，主要是在艾滋病毒/艾滋病中导致危及生命的脑膜脑炎 病人。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 有理由消除与隐球菌病相关的死亡率。这项建议的目的是 确定CN如何感知温度并将其传输到下游的关键效应器通路 致病性。中心假设是细丝形成的GTP酶，称为隔膜素，提供了一种必要的 HUB蛋白复合体，将温度感知与代偿信号反应和血浆联系起来 膜(PM)动态平衡。据认为，暴露在宿主温度下会导致流动性增加和 PM内的曲率和PM相关的磷脂酰肌醇4，5-二磷酸(PIP2)的升高 基于PIP2结合导致PM中Septins的丰富，这在CN适应 寄主温度：它维持质膜的动态平衡，并通过磷脂酶C(PLC)促进胁迫信号传递，以及 蛋白激酶C(PKC)途径。为了支持这一假设，对重组Septins或 利用模式生物的研究表明，毛细管膜蛋白通过其倾向于识别膜的弯曲 绑定PIP2。重要的是，重组分隔素可以防止温度引起的脂双层组成的变化。 支持这一应用的初步数据是：1)CN PIP2水平在37℃时增加。2)Septins与 当CN移动到37℃时PM。3)Septin缺陷突变体表现出PM通透性增加和 对扰乱首相的药物的敏感性已发表的和初步的数据也证明了表型相似性 在Septin缺陷和PLC信号缺陷突变体之间。核心假设将通过以下方式进行检验 追求两个目标。目的：用TIRF显微镜研究PM时Septin组装的动力学。 遗传和药理学方法将决定是否在PM富含Septin复合体 基于PIP2水平的增加。一种新的光可切换等位基因CDc42(参与Septin的GTPase) 组装)将有助于建立作用于隔膜复合体中的CdC42下游的效应器。分隔符 缺乏两亲性螺旋(AH)的突变体将被用来测试AH在招募Septin复合体以 首相目的：葡聚糖复合体对PM脂组成、PM生物物理性质、应激的影响 依赖于PIP2的反应信号以及在动物感染模型中的发病机制将被确定。这个 拟议的研究具有重要意义，因为它将阐明一种新的机制，通过这种机制，七叶皂苷可以发挥作用 感受高温并调节PM依赖的应激反应信号通路 慢性肾炎的发病机制。结果将是更好地理解CN如何适应寄主温度和一个新的 在此基础上为艾滋病毒/艾滋病患者开发改进的抗隐球菌药疗法。