Lipid flippase in echinocandin drug resistance in Cryptococcus neoformans

摘要 隐球菌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。治疗 治疗隐球菌病的选择有限。目前可用的抗真菌药物要么是剧毒的(多烯)，要么是 发挥抑菌作用(三氮唑类)，需要长期的治疗方案，并为 出现抗药性。第三种主要的抗真菌药物，棘球菌素，显示出低毒和 对其他几种流行的真菌病原体有杀菌作用。然而，隐球菌对 棘球菌素和这种耐药性的机制尚不清楚。我们最近报告说，脂肪损失 Flippase，负责维持膜脂双层和正常膜的不对称性的酶 胞内囊泡运输，使新生葡萄球菌对卡泊芬净敏感，卡泊芬净是棘球绦虫类药物，如 以及几种三氮唑。我们还发现，脂质翻转酶对小鼠的毒力是必不可少的。 隐球菌病和致敏新生葡萄球菌对巨噬细胞的杀伤作用，提示这可能是一种新的 抗真菌药物靶标。在这个项目中，我们打算破译隐球菌脂翻转酶的作用机制。 对棘球绦虫的耐药性进行原理验证研究，以抑制新生葡萄球菌的翻转酶功能。 在第一个目标中，我们将测试两个相关的、非互斥的假设，即关于脂质翻转酶在 耐药性：(1)脂转运酶的缺失改变了膜的结构，如PS在膜上的分布， 促进卡泊芬净与其靶向β-1，3-D-葡聚糖合成酶(FKS1)的相互作用，以及(2)在 某些耐药途径，如钙调神经磷酸酶途径，会受到影响。 破坏细胞内钙稳态，并通过药物诱导死亡。我们将通过以下方式检验这些假设： 一系列细胞、分子、生化和遗传方法。在第二个目标中，我们建议发展 抗脂翻转酶胞外环的抗体Fab片段，这是翻转酶功能所必需的， 并测试其使新生葡萄球菌对抗真菌药物敏感和被巨噬细胞杀死的能力。所在地区 这种基于抗体的方法靶向的脂质翻转酶与其人类对应物具有较低的序列同源性， 我们的初步研究表明，针对该区域产生的抗体是真菌特异性的。的成功之处 这项研究将有助于更好地理解脂转移酶介导的新生葡萄球菌的耐药性。 可以帮助扩大棘球菌素药物对隐球菌和其他耐药真菌病原体的使用。 此外，Flippase抑制抗体的产生将提供一个有价值的研究工具，并可能导致 新的联合治疗方法的未来发展。最后，成功研制了抗体-- 碱基抑制剂可能为针对其他膜蛋白的研究和药物开发开辟一条新的途径 在真菌和细菌中。

Abstract Cryptococcosis is a deadly fungal disease that accounts for over 15% HIV/AIDS related deaths. Treatment options for cryptococcosis are limited. Currently available antifungal drugs are either highly toxic (polyenes) or exert a fungistatic effect (triazoles), necessitating long treatment regimens and leaving open the avenue for emergence of drug resistance. The third major antifungal drug class, the echinocandins, show low toxicity and are fungicidal against several other prevalent fungal pathogens. However, Cryptococci are resistant to echinocandins and the mechanisms of this resistance remain unknown. We recently reported that loss of lipid flippase, the enzyme responsible for maintaining the asymmetry of membrane lipid bilayers and normal intracellular vesicle trafficking, sensitizes C. neoformans to caspofungin, a drug of the echinocandin class, as well as to several triazoles. We also found that lipid flippase was essential for virulence in a murine model of cryptococcosis and sensitized C. neoformans to killing by macrophages, suggesting that it may be a novel antifungal drug target. In this project, we propose to decipher the mechanism of lipid flippase in cryptococcal echinocandin resistance and to conduct proof-of-principle studies inhibiting flippase function in C. neoformans. In the first Aim, we will test two related, non-mutually exclusive hypotheses regarding the role of lipid flippase in drug resistance: (1) that loss of lipid flippase changes membrane structure, e.g. PS distribution on membrane, to promote the interaction of caspofungin with its target β-1,3-D-glucan synthase (Fks1), and (2) that in the absence of lipid flippase certain drug resistance pathways, such as calcineurin pathway are compromised, disrupting cellular calcium homeostasis and inducing killing by drugs. We will test these hypotheses by employing a host of cellular, molecular, biochemical, and genetic approaches. In the second Aim, we propose to develop an antibody Fab fragment against the extracellular loop of lipid flippase, which is essential for flippase function, and to test its ability to sensitize C. neoformans to antifungal drugs and to killing by macrophages. The region of lipid flippase 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. The success of this study will lead to a better understanding of lipid flippase mediated drug resistance in C. neoformans, which could help expand the use of echinocandin drugs against Cryptococci and other resistant fungal pathogens. Furthermore, generation of flippase inhibitory antibodies will provide a valuable research tool and may lead to future development of novel combination therapy approaches. Finally, successful development of antibody- based inhibitors could open a new avenue of research and drug development against other membrane proteins in fungi and bacteria.