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于疾病。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类防御素（hBDs）减少引起的。组抑素是由主要唾液腺分泌的富含组氨酸的阳离子蛋白家族，对唾液的抗真菌活性有重要贡献。组蛋白5 （hst5）在家族中具有最高的杀真菌活性，通过引起白色念珠菌胞内离子和核苷酸的选择性释放来杀死酵母。HBDs与hst5在杀真菌途径上有一些相似之处，但其毒性的确切机制尚不清楚。hst5的细胞毒性首先与细胞壁结合，然后进行细胞质运输，破坏细胞内离子运输并引起高渗应激。我们已经确定了细胞壁定位的白色念珠菌Ssa2蛋白，这是一种辅助细胞内运输的伴侣蛋白，但其运输机制的分子特性尚不清楚。我们的初步数据表明，渗透和内吞作用参与肽易位。Hst 5或其他相关阳离子肽递送的一个重要障碍是细胞外盐破坏了Hst 5与酵母细胞壁表面的初始结合。本研究的目的是设计盐不敏感肽，使其能够有效地转运到细胞中，为开发基于肽的假丝酵母菌病治疗剂奠定基础。因此，我们的目标是确定在高盐条件下发挥作用的hst5的最小结构域，通过螺旋盖结构基序增强其耐盐性，并验证它们是否保持最佳的运输特性。对Hst 5处理的白色念珠菌细胞的芯片分析表明，通过Hog1 MAPKinase通路的渗透应激反应是细胞从Hst 5毒性中恢复的重要机制。口腔环境中暴露于生理水平抗真菌肽的念珠菌细胞可能通过激活Hog1p产生耐药性。我们的总体假设是，确定应激反应途径和启动信号的传感器将指导克服念珠菌适应性抵抗的策略。拟议研究的目的是确定真菌细胞摄取肽和随后调节Hst 5和hBD毒性的适应性反应所需的关键因素。这种方法将支持我们的长期目标，即开发基于肽的替代疗法来治疗口腔念珠菌病，目前仅限于一小部分抗真菌药物。

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.