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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.

摘要