Role of phospholipids in antifungal drug resistance in Cryptococcus neoformans

磷脂在新型隐球菌抗真菌药物耐药性中的作用

基本信息

批准号：
10389392
负责人：
Chaoyang Xue
金额：
$ 59.53万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10389392
关键词：
3-Dimensional AIDS/HIV problem ATP phosphohydrolase Acquired Immunodeficiency Syndrome Animal Model Antibodies Antifungal Agents Antifungal Therapy Bacteria Binding Biological Calcium Calcium Channel Calcium Signaling Caspofungin Cell Death Cell Membrane Structures Cell membrane Cell physiology Cell surface Cells Cellular Membrane Cessation of life Chelating Agents Combined Modality Therapy Cryo-electron tomography Cryptococcosis Cryptococcus Cryptococcus gattii Cryptococcus neoformans Data Development Disease Drug Targeting Drug resistance Drug resistance pathway Electron Microscope Enzymes Epitopes Exhibits Fab Immunoglobulins Fungal Drug Resistance Future Generations Genetic study Goals Homeostasis Human Immunoglobulin Fragments Immunoprecipitation Impairment In Vitro Industrial fungicide Infection Ions Knowledge Lead Lipid Bilayers Lipids Liposomes Mass Spectrum Analysis Mediating Membrane Membrane Proteins Methods Mission Modeling Molecular Molecular Weight Monoclonal Antibodies Mus Mycoses Outcome Peptides Phagocytosis Pharmaceutical Preparations Phosphatidylserines Phospholipids Polyenes Proteins Resistance Role Scanning Sequence Homology Siblings Structure Surface Testing Therapeutic Toxic effect Treatment Protocols Triazoles United States National Institutes of Health Vesicle Virulence base cell growth regulation disorder control disorder prevention drug development drug distribution drug sensitivity echinocandin resistance efficacious treatment exportin 1 protein forward genetics fungus glucan synthase human pathogen in vivo inhibitor macrophage mouse model mutant new therapeutic target novel novel therapeutics overexpression pathogenic fungus polyclonal antibody pre-clinical research and development resistance mechanism success trafficking treatment strategy uptake yeast two hybrid system

项目摘要

Abstract Cryptococcus neoformans and its sibling species C. gattii cause Cryptococcosis, a deadly fungal disease that accounts for over 15% of HIV/AIDS related deaths. Treatment options for cryptococcosis remain limited to two drug classes that are either highly toxic (polyenes) or exert a fungistatic effect (triazoles) that necessitate long treatment regimens and can induce drug resistance. The third antifungal drug class, echinocandins, shows low toxicity and is fungicidal against some prevalent fungal pathogens. However, Cryptococcus species are resistant to echinocandins through an unknown resistance mechanism. We found that loss of Cdc50, the regulatory subunit of lipid flippase, an enzyme that maintains asymmetry of the membrane lipid bilayers and regulates intracellular vesicle trafficking, sensitizes C. neoformans to the echinocandin drug caspofungin and several triazoles. We further showed that the cdc50∆ mutant abolishes lipid flippase activity. We also found that this Cdc50-mediated echinocandin resistance requires a mechanosensitive calcium channel protein, Crm1, which modulates intracellular calcium homeostasis. Strikingly, we discovered that lipid flippase function is essential for virulence in a murine model of cryptococcosis, suggesting that lipid flippase may be a novel antifungal drug target. In this project, our goals are to determine how lipid flippase mediates cryptococcal echinocandin resistance, and to conduct proof-of-principle studies of antibody-based inhibitors targeting flippase function as novel therapeutics for Cryptococcus infections. We hypothesize that C. neoformans has a unique plasma membrane structure and that loss of lipid flippase alters that structure to promote the interaction of caspofungin with its target and compromises fungal drug resistance mechanisms. We propose three Aims to test our hypothesis. In Aim 1, we will elucidate how loss of Cdc50 changes membrane structure to promote the interaction of caspofungin with its membrane target β-1,3-D-glucan synthase (Fks1). Aim 2 will identify the downstream drug resistance pathways that are compromised by the absence of Cdc50, which disrupts intracellular calcium homeostasis and promotes cell death. In Aim 3, we will develop an antibody Fab fragment and a stable peptide against the exoplasmic loop of Cdc50, which is essential for flippase function. We will validate how inhibitors sensitize C. neoformans to antifungal drugs and macrophage killing in vitro and in vivo in animal models. The region of Cdc50 targeted by this antibody-based approach has low sequence homology to its human counterpart, and our preliminary studies showed that an antibody raised against this region is fungal- specific, reducing the chance of off-target effects. The impact of this study to elucidate the mechanisms underlying lipid flippase mediated drug resistance in C. neoformans will be developing strategies for exploiting echinocandin drugs to effectively treat Cryptococci and other resistant fungal pathogens. Our successful development of antibody-based inhibitors will establish a new avenue of research and drug development against other membrane proteins in fungi and bacteria.

抽象的新型隐球菌及其同胞种格特隐球菌引起隐球菌病，这是一种致命的真菌病，占艾滋病毒/艾滋病相关死亡人数的 15% 以上，隐球菌病的治疗选择仍然仅限于两种。具有剧毒（多烯）或具有抑制真菌作用（三唑）的药物类别，需要长期使用第三类抗真菌药物，棘白菌素，显示出较低的治疗方案。毒性并对一些常见的真菌病原体具有杀菌作用，但隐球菌属具有抗药性。我们发现，棘白菌素通过一种未知的耐药机制失去了调节作用。脂质翻转酶的亚基，一种维持膜脂质双层不对称性并调节的酶细胞内囊泡运输，使新型隐球菌对棘白菌素药物卡泊芬净和几种药物敏感我们进一步表明 cdc50Δ 突变体消除了脂质翻转酶活性。 Cdc50 介导的棘白菌素耐药性需要机械敏感性钙通道蛋白 Crm1，该蛋白引人注目的是，我们发现脂质翻转酶功能对于调节细胞内钙稳态至关重要。在隐球菌病鼠模型中的毒力表明脂质翻转酶可能是一种新型抗真菌药物在这个项目中，我们的目标是确定脂质翻转酶如何介导隐球菌棘白菌素。耐药性，并对靶向翻转酶功能的基于抗体的抑制剂进行原理验证研究我们勇敢地承认新型隐球菌具有独特的血浆。膜结构和脂质翻转酶的损失改变了该结构以促进卡泊芬净的相互作用我们提出了三个目标来测试我们的目标。在目标 1 中，我们将阐明 Cdc50 的损失如何改变膜结构以促进卡泊芬净与其膜靶点 β-1,3-D-葡聚糖合酶 (Fks1) 的相互作用将鉴定出下游耐药途径因 Cdc50 的缺失而受到损害，从而破坏了细胞内钙稳态并促进细胞死亡在目标 3 中，我们将开发抗体 Fab 片段。以及针对 Cdc50 外质环的稳定肽，这对于翻转酶功能至关重要。在体外和体内验证抑制剂如何使新型隐球菌对抗真菌药物和巨噬细胞杀伤敏感这种基于抗体的方法所靶向的 Cdc50 区域与动物模型具有较低的序列同源性。它的人类对应物，我们的初步研究表明针对该区域产生的抗体是真菌- 具体来说，减少了脱靶效应的机会。这项研究阐明了其机制。潜在的脂质翻转酶介导的新型隐球菌耐药性将制定利用策略我们成功地使用棘白菌素药物来有效治疗隐球菌和其他耐药真菌病原体。基于抗体的抑制剂的开发将为针对癌症的研究和药物开发开辟新途径真菌和细菌中的其他膜蛋白。