CANDIDACIDAL MECHANISMS OF SALIVARY HISTATINS

描述(申请人提供)：由白色念珠菌引起的口咽念珠菌病在接受癌症化疗的患者、艾滋病患者、糖尿病患者或早产儿中很常见。人唾液组织蛋白5(HST 5)是一种对人体无毒的有效杀菌蛋白，具有杀灭耐唑念珠菌的能力。我们以前的研究已经证明，HST-5通过一个复杂的多机制过程发挥抗真菌活性。毒素活性是通过与白念珠菌细胞膜上的70 kDa蛋白结合而启动的，我们最近发现Ssa1是HSP70家族中的一个成员，参与蛋白质的结合和跨细胞膜的运输。在细胞内转位后，HST5可能通过Trk1破坏膜离子梯度，导致体积调节中断和G1细胞周期停滞。然而，在我们对这一进程的理解中仍然存在许多差距。我们不知道HST 5与Ssa1蛋白结合的关键要求。确定HST-5与SSA蛋白的精确结合部位是设计针对酵母细胞表面受体的抗真菌毒性蛋白的关键。此外，我们还不知道参与HST 5破坏离子稳态的酵母通道的身份，也不知道最终导致细胞周期停滞的全球变化。为了更全面地了解这些过程，我们建议1)在体外鉴定Ssa1p中的HST 5结合结构域，2)评估在体内HST 5功能所需的Ssa1/2p的作用，3)确定主要的酵母钾通道Trk1p在HST 5杀伤中的作用，以及4)利用全基因组白念珠菌微阵列分析HST 5改变的酵母基因转录本，特别是细胞周期调控基因。了解HST-5诱导的杀伤机制及其对酵母细胞相对于人类细胞的特异性的本质将有助于未来改进的抗真菌药物的开发。

DESCRIPTION (provided by applicant): Oropharyngeal candidiasis caused by Candida albicans is prevalent in patients receiving cancer chemotherapy, AIDS patients, diabetics, or premature infants. Human salivary Histatin 5 (Hst 5) is a potent fungicidal protein that lacks toxicity to humans and has the ability to kill azole-resistant Candida strains. Our previous studies have demonstrated that Hst 5 exerts antifungal activity through a complex multi-mechanistic process. Toxic activity is initiated by binding to a C. albicans 70 kDa protein in the cell envelope that we have recently identified as Ssa1, a member of the HSP70 family involved in binding and transport of proteins across cell membranes. Following intracellular translocation, Hst 5 disrupts membrane ion gradients, perhaps through Trk1, resulting in disruption of volume regulation and G1 cell cycle arrest. However, many gaps remain in our understanding of this process. We do not know the critical requirements for Hst 5 binding with Ssa1 proteins. Delineation of the precise binding sites of Hst 5 with Ssa protein are crucial in design of antifungal toxic proteins targeted to the yeast cell surface receptors. In addition, we do not yet know the identity of yeast channels that participate in ion homeostasis disrupted by Hst 5, nor do we understand the global changes that occur ultimately leading to cell cycle arrest. To more fully understand these processes, we propose to 1) Identify the Hst 5 binding domain in Ssa1p in vitro 2) Assess the role of Ssa1/2p required for Hst 5 function in vivo, 3) Determine the role of the major yeast potassium channel, Trk1p, in Hst 5 killing and 4) Identify yeast gene transcripts, especially cell cycle regulatory genes, altered by Hst 5 using full genome C. albicans microarray analysis. An understanding of the mechanism of Hst 5-induced killing and the nature of its specificity for yeast cells over human cells will contribute to future development of improved antifungal drugs.