CANDIDACIDAL MECHANISMS OF SALIVARY HISTATINS

唾液组氨酸的抗念珠菌机制

• 批准号: 2131541
• 负责人: Mira Edgerton
• 金额: $ 10.3万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2131541
• 关键词: Candida albicans; antifungal agents; calcium flux; calorimetry; candidiasis; cell membrane; chemical structure function; chemical substitution; circular dichroism; drug screening /evaluation; ergosterol; glycoproteins; high performance liquid chromatography; histidine; human tissue; intermolecular interaction; liposomes; membrane activity; membrane lipids; protein purification; protein structure function; protoplast /spheroplast; saliva; synthetic peptide; tissue /cell culture

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.

唾液组胺素是一个家族，至少有12个多肽（7-38个氨基酸 酸），其已被证明是有效的杀念珠菌分子， 并且是酵母细胞生长和萌发的活性抑制剂。 序列和构象数据可用于 组胺素家族和所需分子的最小肽部分 已经确定了完全的杀念珠菌活性。 但不同于 大多数其他作用机制良好的抗真菌药物 特征，很少有人知道的分子基础 组蛋白的抗真菌活性。 更完整地理解 需要这些唾液分子的分子作用机制， 为了设计将组胺素递送至选定的口腔部位的方法， （与固体表面连接或与口腔粘膜相关），以及 设计功能增强的合成肽。 与咪唑类抗真菌药一样，组蛋白似乎影响念珠菌和真菌。 细胞膜功能以及麦角甾醇（一种关键的真菌甾醇） 生物合成 高度保守的杀念珠菌基因中的组氨酸基团 结构域可以提供对接或细胞内易位功能。 这些功能域分配的有效性和特定的 组蛋白的作用机制将使用以下方法进行研究 接近。 四种模型分子组成，如果杀念珠菌结构域 (A16)A16和N-末端结构域（组胺素5）， A16和C-末端结构域（组胺素4），以及A16和C-和N- 末端结构域（组蛋白3）将比较它们的能力， 影响细胞膜完整性、麦角固醇生物合成和抑制 的附着力。 此外，还提供了具有氨基酸的模型肽， 在活性位点处的取代和受约束的螺旋肽将 进行杀念珠菌活性测试。 具体而言，膜效应将 通过比较这些模型肽释放的能力来检查 来自白色念珠菌全细胞和原生质球（细胞）细胞内染料 无壁）;和体外通过分析脂质体-肽相互作用。 体外模型膜相互作用将使用三种 补充测定：a）组胺素诱导的脂质体破坏， 通过释放包封的染料; B）测量 脂质体，使用差示扫描量热法（DSC）;和 通过环状测量的组胺素肽的诱导构象变化 二向色性（CD）。 放射性标记前体的麦角固醇进入 麦角甾醇治疗后与四个模型组胺和氨基 酸取代肽。 最后，抑制C. 白色念珠菌唾液涂层聚（甲基丙烯酸甲酯）（PMMA）， 将评估用亚致死剂量的组蛋白预处理细胞。