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 隐球菌病的选择是有限的。目前可用的抗真菌药物要么是高毒性（多烯）， 发挥真菌抑制作用（三唑类），需要长时间的治疗方案，并留下开放的途径， 耐药性的出现。第三类主要的抗真菌药物棘白菌素显示出低毒性， 对其它几种常见的真菌病原体具有杀真菌作用。然而，隐球菌对 棘白菌素和这种耐药性的机制仍然未知。我们最近报道， 翻转酶，该酶负责维持膜脂双层的不对称性和正常的 胞内小泡运输，使C.卡泊芬净是棘白菌素类药物， 以及几种三唑。我们还发现，脂质翻转酶在小鼠模型中的毒力是必不可少的。 隐球菌病和致敏C.这可能是一种新的， 抗真菌药物靶点。在这个项目中，我们打算破译隐球菌中脂质翻转酶的机制， 棘白菌素抗性，并进行抑制C.新人类 在第一个目标中，我们将测试两个相关的，不相互排斥的假设，关于脂质翻转酶在 耐药性：（1）脂质翻转酶的丧失改变了膜结构，如膜上的PS分布， 促进卡泊芬净与其靶向β-1，3-D-葡聚糖合酶（Fks 1）的相互作用，以及（2） 脂质翻转酶的缺乏会损害某些药物抗性途径，例如钙调神经磷酸酶途径， 破坏细胞钙稳态并诱导药物杀伤。我们将通过使用 细胞、分子、生物化学和遗传学方法的主机。在第二个目标中，我们建议开发 抗脂质翻转酶胞外环的抗体Fab片段，其对于翻转酶功能是必需的， 并检测其致敏C.抗真菌药物和巨噬细胞的杀伤作用。的区域 这种基于抗体的方法靶向的脂质翻转酶与其人对应物具有低序列同源性， 我们的初步研究表明，针对这一区域产生的抗体是真菌特异性的。的成功 本研究将有助于更好地了解脂质翻转酶介导的C.新形式， 可以帮助扩大棘白菌素药物对隐球菌和其他耐药真菌病原体的使用。 此外，翻转酶抑制性抗体的产生将提供有价值的研究工具，并可能导致 新的联合治疗方法的未来发展。最后，抗体研制成功- 的抑制剂可以开辟一条新的研究和药物开发的途径，针对其他膜蛋白 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.