Candidacidal Mechanisms of Salivary Histatins

描述（由申请人提供）：口咽念珠菌病或鹅口疮是免疫功能低下患者以及老年人和幼儿的常见疾病。口腔中白色念珠菌的过度生长是由唾液减少或粘膜抗真菌肽如人防御素（hBD）减少引起的。组蛋白是由唾液腺分泌的一类富含组氨酸的阳离子蛋白，对唾液的抗真菌活性有重要作用。组蛋白5（Hst 5）是该家族中具有最高杀真菌活性的蛋白，它通过选择性地释放胞内离子和C.白色念珠菌。HBDs与Hst 5在杀真菌途径上有一些相似之处，但其确切的毒性机制尚不清楚。Hst 5的细胞毒性是通过与细胞壁结合，然后通过胞质转运而引发的，在胞质转运中，Hst 5破坏细胞内离子转运并引起高渗应激。我们已经确定了细胞壁定位C。白念珠菌Ssa 2蛋白是协助细胞内转运的伴侣蛋白，但转运机制的分子身份尚不清楚。我们的初步数据表明，渗透酶和内吞作用参与肽移位。传递Hst 5或其它相关阳离子肽的显著障碍是胞外盐破坏Hst 5与酵母细胞壁表面的初始结合。该提议的目的是设计盐不敏感肽，其被有效转运到细胞中作为开发用于念珠菌病的基于肽的治疗剂的基础。因此，我们的目标是确定最小域的HST 5功能在高盐条件下，提高其耐盐性与螺旋帽图案，并验证它们保留最佳的运输性能。基因芯片分析Hst 5处理的C.白念珠菌细胞显示，通过Hog1 MAP激酶途径的渗透应激反应是细胞从Hst 5毒性恢复的重要机制。在口腔环境中暴露于生理水平的抗真菌肽的牙周细胞可能通过Hog1p的激活产生耐药性。我们的总体假设是，定义应激反应途径和启动信号传导的传感器将指导克服植物适应性抗性的策略。拟议的研究的目的是确定真菌细胞摄取肽和随后的适应性反应，调节HST 5和hBD毒性所需的关键要素。这种方法将支持我们的长期目标，即开发用于治疗口腔念珠菌病的替代性基于肽的疗法，目前仅限于一小组抗真菌药物。 公共卫生相关性：免疫功能低下的患者以及老年人和幼儿对口咽念珠菌病或口腔鹅口疮有很高的易感性。本提案的目的是设计选择性靶向酵母细胞的盐不敏感肽作为念珠菌病的口服治疗剂。

DESCRIPTION (provided by applicant): Oropharyngeal candidiasis, or thrush, is a frequent disease among immunocompromised patients as well as in elderly people and young children. Overgrowth of Candida albicans in the oral cavity is caused by decreases in saliva or reduction in mucosal antifungal peptides such as human -defensins (hBDs). Histatins are a family of histidine-rich cationic proteins secreted by major salivary glands that contribute significantly to the antifungal activity of saliva. Histatin 5 (Hst 5) has the highest fungicidal activity of the family, and kills yeast by causing selective release of intracellular ions and nucleotides from C. albicans. HBDs share some similarities in fungicidal pathways with Hst 5, but their precise mechanism of toxicity is not known. Cytotoxicity of Hst 5 is initiated by binding to the cell wall followed by cytosolic transport where it disrupts intracellular ion tranport and causes hyperosmotic stress. We have identified cell wall localized C. albicans Ssa2 proteins that are chaperone proteins assisting intracelluar transport, but the molecular identity of the transport mechanism is not known. Our preliminary data show that permeases and endocytosis are involved in peptide translocation. A significant barrier to delivery of Hst 5 or other related cationic peptides is disruption of initial binding of Hst 5 to the yeast cell wall surface by extracellular salts. Objectives of this proposal are design of salt-insensitive peptides which are efficiently transported into the cell as a basis for development of peptide- based therapeutic agents for candidiasis. Therefore, our aims are to identify minimal domains of Hst 5 that function under high salt conditions, enhance their salt resistance with helix-capping motifs, and verify that they retain optimal transport properties. Microarray analyses of Hst 5 treated C. albicans cells show that osmotic stress response by Hog1 MAPKinase pathway is an important mechanism for recovery of cells from Hst 5 toxicity. Candidal cells exposed to physiological levels of antifungal peptides in the oral environment may develop resistance through activation of Hog1p. Our overall hypothesis is that defining stress response pathways and the sensors that initiate signaling will guide strategies to overcome Candidal adaptive resistance. The objectives of the proposed studies are to identify key elements required for fungal cell uptake of peptides and subsequent adaptive responses that modulate Hst 5 and hBD toxicity. This approach will support our long-range goal to develop alternative peptide- based therapies for treatment of oral candidiasis, which is currently limited to a small group of antifungal drugs. PUBLIC HEALTH RELEVANCE: Immunocompromised patients as well as elderly people and young children have a high susceptibility to oropharyngeal candidiasis or oral thrush. Objectives of this proposal are to design salt-insensitive peptides that selectively target yeast cells as oral therapeutic agents for candidiasis.