Role of phospholipids in antifungal drug resistance in Cryptococcus neoformans

摘要 新生隐球菌及其兄弟种加蒂亚氏隐球菌会引起隐球菌病，这是一种致命的真菌疾病， 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限于两种 剧毒类药物(多烯类)或具有抑菌作用的药物类(三唑类)，需要长时间 治疗方案和可能会导致耐药性。第三类抗真菌药物棘球菌素类药物显示较低 对一些流行的真菌病原体有毒性和杀菌作用。然而，隐球菌种是有抗药性的 通过一种未知的抗性机制对棘球菌素类药物产生抗药性。我们发现，监管机构CDC50的损失 脂翻转酶的亚基，维持膜脂双层的不对称性并调节 胞内囊泡运输，使新生葡萄球菌对棘球绦虫药物卡泊芬净和几种 三氮唑。我们进一步证明，CDC50∆突变体可使脂质翻转酶活性丧失。我们还发现这一点 CDC50介导的棘球绦虫耐药需要一种机械敏感的钙通道蛋白CRM1，它 调节细胞内钙稳态。令人惊讶的是，我们发现脂质翻转酶的功能对于 隐球菌病小鼠模型的毒力，提示脂翻转酶可能是一种新的抗真菌药物 目标。在这个项目中，我们的目标是确定脂转运酶如何介导隐球菌棘球绦虫粘附素 抗药性，并进行针对Flippase功能的基于抗体的抑制剂的原理证明研究 治疗隐球菌感染的新疗法。我们假设新生葡萄球菌有一种独特的血浆 膜结构和脂翻转酶丢失改变该结构以促进卡泊芬净的相互作用 与其靶点和妥协的真菌耐药性机制。我们提出三个目标来测试我们的 假设。在目标1中，我们将阐明cdc50的缺失是如何改变膜结构以促进 卡泊芬净与其膜靶标β-1，3-D-葡聚糖合成酶的相互作用。目标2将确定 下游耐药途径因缺乏CDC50而受损，从而破坏 细胞内钙稳态并促进细胞死亡。在目标3中，我们将开发一种抗体Fab片段 以及针对CDC50外质环的稳定多肽，这是Flippase功能所必需的。我们会 验证抑制剂如何在体外和体内增敏新生葡萄球菌对抗真菌药物和巨噬细胞杀伤作用 动物模型。这种基于抗体的方法靶向的cdc50区域与 我们的初步研究表明，针对这一区域产生的抗体是真菌- 具体地说，减少偏离目标的影响的机会。这项研究对阐明其作用机制的影响 新生假单胞菌潜在的脂转移酶介导的耐药性将被开发利用的策略 棘球菌素类药物可有效治疗隐球菌和其他耐药真菌病原体。我们的成功 基于抗体的抑制剂的开发将为研究和药物开发开辟一条新的途径 真菌和细菌中的其他膜蛋白。

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.