CANDIDACIDAL MECHANISMS OF SALIVARY HISTATINS

唾液组抑素是一个至少由12个多肽(7-38个氨基酸)组成的家族 酸)，已被证明是有效的杀菌分子，如 也是酵母细胞生长和萌发的活性抑制剂。 序列和构象数据可供 组蛋白家族和所需分子的最小多肽部分 已经确定了完全杀灭假丝酵母菌的活性。然而，不同于 作用机制良好的大多数其他抗真菌药物 其特征是，对其分子基础知之甚少 他汀类药物的抗真菌活性。更全面地了解 这些唾液分子的分子作用机制在 设计组抑素在选定口腔部位的给药方法 (连接到固体表面或与口腔粘膜相关)，以及 如设计具有增强功能的合成肽。 像咪唑类抗真菌药一样，组他汀类药物似乎对两种念珠菌都有影响 细胞膜的功能与麦角甾醇(一种关键的真菌甾醇)一样 生物合成。高度保守的念珠菌中的组氨酸基团 结构域可以提供对接或细胞内转位功能。 这些功能域的分配的有效性和特定的 组他汀类药物的作用机制将使用以下方法进行研究 接近了。由假丝酵母菌结构域组成的四个模型分子 (A16)(GYKRKFHEKHHSHRGY)单独，A16和N-末端结构域(组蛋白5)， A16和C-末端结构域(组蛋白4)，以及A16和C-和N- 将比较末端结构域(组蛋白3)的能力 影响细胞膜完整性、麦角固醇生物合成和抑制 粘附力。此外，一种含有氨基酸的模型肽 活性位点上的取代和受限制的螺旋多肽将 接受杀菌活性检测。具体地说，膜效应将 通过比较这些模型多肽的释放能力来检验 白色念珠菌全细胞和球体(细胞)的胞内染料 无壁)；以及通过脂质体-多肽相互作用的分析在体外。 在体外模型膜相互作用将测试三个 补充分析：a)组蛋白诱导的脂质体破坏 通过释放包裹的染料；b)测量流动性的变化 采用差示扫描量热法(DSC)的脂质体；以及 环法测定组蛋白多肽的诱导构象变化 二向色性(CD)。麦角甾醇对放射性标记前体的抑制作用 四种模型组素和氨基治疗后的麦角甾醇 酸取代多肽。最后，对C. 白色念珠菌对唾液包被聚甲基丙烯酸甲酯(PMMA)的影响 使用亚致死剂量的组他汀类药物对细胞进行预处理将进行评估。

Salivary histatins are a family of at least 12 polypeptide (7-38 amino acids) which have been shown to be effective candidacidal molecules as well as being active inhibitors of yeast cell growth and germination. Sequence and conformational data is available for the members of the histatin family and the minimal peptide portion of the molecule required for complete candidacidal activity has been identified. However, unlike most other antimycotic drugs where the mechanism of action is well characterized, very little is known about the molecular basis of histatins' antifungal activity. A more complete understanding of the molecular mechanism of action of these salivary molecules is required in order to design methods of delivery of histatins to selected oral sites (linkage to solid surfaces or associated with the oral mucosa), as well as designing synthetic peptides with enhanced function. Like the imidazole antimycotic, histatins appear to affect both candidal cell membrane function as well as ergosterol (a key fungal sterol) biosynthesis. Histidine groups in the highly conserved candidacidal domains may provide docking or intracellular translocation functions. The validity of assignment of these functional domains and the specific mechanisms of action of histatins will examined using the following approaches. Four model molecules consisting if the candidacidal domain (A16) (GYKRKFHEKHHSHRGY) alone, A16 and N-terminal domains (histatin 5), A16 and C-terminal domains (histatin 4), and A16 and both C- and N- terminal domains (histatin 3) will be compared in their abilities to affect cell membrane integrity, ergosterol biosynthesis and inhibition of adhesion. In addition, a model peptide having amino acid substitutions at the active site and a constrained helical peptide will be tested for candidacidal activity. Specifically, membrane effects will be examined by comparing the ability of these model peptides to release intracellular dye from Candida albicans whole cells and spheroplast (cell wall free); and in vitro by analysis of liposome-peptide interactions. In vitro model membrane interactions will be tested with three complementary assays: a) histatin induced liposome disruption as measured by release of encapsulated dye; b) measurement of change in fluidity of liposomes using differential scanning calorimetry (DSC); and c) liposome induced conformational change of histatin peptides measured by circular dichroism (CD). Inhibition of ergosterol of radiolabeled precursors into ergosterol following treatment with the four model histatin and amino acid substituted peptides. And finally, inhibition of adhesion of C. albicans to saliva coated poly (methyl methacrylate) (PMMA) by pretreating cells with sublethal doses of histatins will be assessed.