Microbicidal Salivary Histidine-Rich Proteins

杀菌唾液富含组氨酸的蛋白质

• 批准号: 6833489
• 负责人: Frank G Oppenheim
• 金额: $ 40.75万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833489
• 关键词: Actinobacillus actinomycetemcomitans; Bacteroides gingivalis; Candida albicans; Streptococcus pyogenes; antibacterial agents; antifungal agents; antioxidants; bacterial proteins; bacterial toxins; capillary electrophoresis; cathepsin B; cathepsin G; cell death; cell membrane; collagenase; flow cytometry; free radical oxygen; fungal proteins; histidine; human tissue; nuclear magnetic resonance spectroscopy; protein structure function; saliva; secretory protein; superoxide dismutase; yeast two hybrid system

Histatins constitute a distinct family of low molecular weight, histidine-rich, cationic salivary proteins which exhibit a broad range of antimicrobial activities. Unlike several other cationic antimicrobial peptides which act in local environments and in close proximity to their site of synthesis, histatins are secreted by both parotid and submandibular glands and are subsequently transported to the oral cavity protecting oral, pharyngeal and esophageal tissues. The long term objective of this project is to understand how histatins, representing an important part of the oral innate host defense system, protect against the multiple potentially adverse effects of microorganisms entering and residing in the oral cavity. The mechanism by which histatins kill Candida albicans, a pathogenic yeast, has not been fully elucidated. It has been shown that histatins, by virtue of their weakly amphipathic nature and reluctance to form helical structures in hydrophobic environments, do not form pore structures in cell membranes. Since histatins are taken up only by metabolically active cells, target mitochondria, inhibit cellular respiration and form reactive oxygen species (ROS), it is likely that the candidacidal activity of the histatins is related to the deleterious effects of ROS on cellular membranes. Aim 1 focuses on the mechanism of action of histatins by identification of: a) the site of inhibition within the respiratory chain, b) the mechanism of ROS formation triggered by histatins and c) ROS induced destabilization of cell membranes by the characterization of nucleotides and proteins/peptides released into the extracellular environment. Another unique feature of histatins is their effect against bacterial virulence factors such as host tissue destroying enzymes and bacterial toxins. Aim 2 is to characterize these "second generation type antibiotic" effects of histatins by investigating the inhibition of several bacterial enzymes, such as the gingipains from Porphyromonas gingivalis, host-derived proteases such as metalloproteinases and the process of neutralization of the leukotoxin released from the periodontal pathogen Actinobacillus actinomycetemcomitans. Aim 3 will assess the structure/function relationships between histatins and their antimicrobial activity using recombinant technologies to construct artificial histatins containing naturally occurring sequences of functional importance, and to generate hybrid molecules exhibiting bi-functional activities. Aim 4 is planned to investigate the functional consequences of the propensity of histatins to form heterotypic complexes employing the molecular approach of the yeast two-hybrid system and the direct biochemical characterization of complexes formed in saliva. Aim 5 will establish the in vivo relationship between histatin levels in whole saliva and the oral microbial profile using the DNA-DNA checkerboard assay providing quantitative information on C. albicans and 80 species of oral bacteria ranging from harmless commensal organisms to periodontal pathogens.

组织蛋白构成一个低分子量的独特家族， 富含组氨酸的阳离子唾液蛋白，表现出广泛的范围 抗菌活性。与其他几种阳离子抗菌肽不同 在当地环境中起作用，并且非常接近其地点 合成，组蛋白被腮腺和下颌腺分泌，以及 随后运输到保护口腔，咽和 食管组织。该项目的长期目标是了解 组织蛋白如何代表口腔先天宿主防御的重要组成部分 系统，防止多种潜在的不利影响 微生物进入和居住在口腔中。该机制 组织蛋白杀死一种致病性酵母白色念珠菌，尚未完全 阐明。已经表明，组织蛋白因其微弱 两亲性和不愿形成疏水性螺旋结构 环境，不会在细胞膜中形成孔结构。自组织蛋白以来 仅由代谢活性细胞（靶线粒体）抑制 细胞呼吸和形成活性氧（ROS），很可能 组织素的候选活性与有害 ROS对细胞膜的影响。 AIM 1专注于行动机制 通过识别：a）抑制部位 呼吸链，b）组织蛋白和 c）ROS通过表征对细胞膜诱导的不稳定 核苷酸和蛋白质/肽释放到细胞外环境中。 组织蛋白的另一个独特特征是它们对细菌毒力的作用 宿主组织破坏酶和细菌毒素等因素。 AIM 2是 为了表征这些“第二代类型的抗生素”效应 通过研究抑制几种细菌酶，例如 牙龈卟啉单胞菌的牙龈，宿主衍生的蛋白酶，例如 金属蛋白酶和释放的白细胞毒素中和过程 从牙周病原体肌动杆菌放线菌。目标3意志 评估组织蛋白及其其结构/功能关系 使用重组技术来构建人工的抗菌活性 组织蛋白包含自然存在的功能重要性序列， 并产生表现出双功能活性的混合分子。目标4是 计划研究倾向的功能后果 组织蛋白采用分子方法形成异型复合物 酵母双杂交系统和直接生化特征 唾液中形成的复合物。 AIM 5将建立体内关系 在整个唾液中的组织蛋白水平与口服微生物剖面之间使用 DNA-DNA棋盘格测定法，提供有关C的定量信息。 白色疾病和80种口服细菌，范围从无害共生 有机体到牙周病原体。