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：
10654524
负责人：
Chaoyang Xue
金额：
$ 56.73万
依托单位：
RUTGERS BIOMEDICAL AND HEALTH SCIENCES
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 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10654524
关键词：
3-Dimensional 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 D Cells 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 HIV/AIDS Homeostasis Human Immunoglobulin Fragments Immunoprecipitation Impairment In Vitro Infection Ions Knowledge Lipid Bilayers Lipids Liposomes Macrophage 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 cell growth regulation disorder control disorder prevention drug development drug distribution drug sensitivity echinocandin resistance efficacious treatment exportin 1 protein forward genetics fungicide fungus glucan synthase human pathogen in vivo inhibitor 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.

抽象的加密环球球菌及其兄弟姐妹种类C. gattii引起加密coscoccussis，一种致命的真菌疾病，是一种致命的真菌疾病占艾滋病毒/艾滋病相关死亡的15％以上。隐球菌病的治疗选择仍然限制为两个剧毒（polyeners）或发挥必要长的拟合作用（三唑）的药物类别治疗方案并可以诱导耐药性。第三个抗真菌药物类Echinocandins显示出低毒性是对某些普遍的真菌病原体的真菌。但是，加密环球具有抗性通过未知的抗性机制来进行eChinocandins。我们发现CDC50的损失是调节性的脂质Flippase的亚基，一种酶，该酶保持膜脂质双层的不对称和调节细胞内囊泡运输，感应新生虫的棘齿药物caspofungin和几个三轮唑。我们进一步表明，Cdc50Δ突变体废除了脂质氟脂酶的活性。我们还发现这个 Cdc50介导的棘齿抗蛋白的耐药需要机械敏感的钙通道蛋白CRM1，该蛋白调节细胞内钙稳态。令人惊讶的是，我们发现脂质Flippase功能对于在鼠的隐孢子虫模型中，病毒表明脂质氟脂酶可能是一种新型的抗真菌药物目标。在这个项目中，我们的目标是确定脂质Flippase如何介导加密甲虫的echinocandin 抗性，并进行基于抗体的抑制剂的原则研究，以Flippase功能为抗体加密环球感染的新疗法。我们假设C. Neoformans具有独特的等离子体膜结构和脂质Flippase的损失会改变结构以促进caspofungin的相互作用其目标并损害了真菌耐药性机制。我们提出了三个目标，以测试我们的假设。在AIM 1中，我们将阐明CDC50的损失如何改变膜结构以促进 Caspofungin与其膜靶标β-1,3-D-葡聚糖合酶（FKS1）的相互作用。 AIM 2将确定由于缺乏Cdc50而损害的下游耐药性途径，这会破坏在AIM 3中，我们将开发一个抗体Fab片段以及针对CDC50的质质环的稳定肽，这对于Flippase功能至关重要。我们将验证抑制剂如何在体外和体内杀死抗真菌药物和巨噬细胞杀死抗真菌药物和巨噬细胞动物模型。这种基于抗体的方法针对的CDC50区域与较低的序列同源性与它的人类对应物和我们的初步研究表明，对该地区提出的抗体是真菌具体，减少了脱靶效应的机会。这项研究阐明机制的影响 Neoformans中的潜在脂质Flippase介导的耐药性将开发用于利用的策略 echinocandin药物可有效治疗加密环球病毒和其他抗性真菌病原体。我们的成功基于抗体的抑制剂的开发将建立针对研究和药物开发的新途径真菌和细菌中的其他膜蛋白。