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.

项目摘要 该提案的长期目标是发现隐孢子球致病性的机制 （CN）着重于CN如何适应宿主温度。 CN是一种真菌病原体，进入肺部并 通过血液传播，在艾滋病毒/艾滋病中引起威胁生命的脑膜脑炎。 患者。当前的抗癌疗法可能具有毁灭性的副作用。那是新的治疗策略 有必要消除与隐球菌病有关的死亡率。该提议的目的是 确定如何通过CN感应温度并传输到下游效应器途径至关重要 致病性。中心假设是形成细丝的GTP-ass称为septins提供了必不可少的 轮毂蛋白复合物将温度灵敏度与补偿信号响应和等离子体联系起来 膜（PM）稳态。有人提出暴露于宿主温度会导致流动性升高和 PM内的曲率和与PM相关的磷脂酰肌醇4,5-双磷酸的升高（PIP2） 基于PIP2结合，在PM上富集了Septins，在CN适应中具有两倍的作用 宿主温度：它维持PM稳态，并通过磷脂酶C（PLC）促进应力信号，并且 蛋白激酶C（PKC）途径。为了支持这一假设，对重组Septins或 采用模型生物的研究表明，Septins通过承诺识别膜曲率 结合PIP2。重要的是，重组SEPTIS可防止温度引起的脂质双层组成的变化。 支持此应用的初步数据是：1）CN PIP2水平在37°C时增加。 2）Septins与 当CN移至37°C时的PM。 3）缺乏SEPTIN的突变体暴露于PM渗透性增加和 对扰动PM的药物的敏感性。已发布和初步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。中心假设将通过 追求两个目标。 AIM1：将使用TIRF显微镜定义SEPTIN组装的动力学。 遗传和药物方法将确定在PM上是否富含Septin复合物 基于PIP2水平的增加。 CDC42的创新光转换等位基因（Septin涉及的GTPase 组装）将有助于建立在Septin复合物形成中CDC42作用的效应子。 9月 缺乏两亲螺旋（AH）的突变体将用于测试AH在招募Septin Complex中的作​​用 下午。 AIM2：Septin复合物对PM脂质组成，PM生物物理特性，应力的影响 响应信号取决于PIP2，将确定动物感染模型中的发病机理。这 拟议的研究很重要，因为它将阐明一种新的机制 感知高温并控制PM依赖性应力响应信号传导途径，至关重要 CN的发病机理。结果将更好地理解CN如何适应宿主温度和新的 在艾滋病毒/艾滋病患者中，基金会发展了改善的抗癌局疗法。