FIMBRIAL ADHERENCE TO LUNG EPITHELIA BY KLEBSIELLA

克雷伯菌对肺上皮细胞的菌毛粘附

• 批准号: 2210174
• 负责人: DOUGLAS B HORNICK
• 金额: $ 7.73万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2210174
• 关键词: Klebsiella; adhesin; alleles; antibacterial antibody; bronchoscopy; cell adhesion; cell transformation; collagen; epitope mapping; gene deletion mutation; gene mutation; human subject; human tissue; laboratory rabbit; lung; mutant; nucleic acid sequence; pilus; protein sequence; respiratory epithelium; site directed mutagenesis; trachea

Research: Attachment the respiratory epithelium is believed to be an early step in the development of pneumonia. Klebsiella produce filamentous organelles called fimbriae which carry adhesive protein subunits called adhesins. This proposal will further characterize the Klebsiella type 3 fimbrial adhesin responsible for adherence to respiratory epithelium. The initial experiments will identify which DNA sequence(s) of the type 3 fimbrial adhesin gene, mrkD, are critical for proper function of the adhesin. The two necessary recombinant plasmids producing transformed bacteria that express nonadhesive type 3 fimbriae (pFK28) and type 3 fimbrial adhesin protein (pFK58) were previously constructed. Hence, in this study, site-specific and random mutations in pFK58 can be produced and the appropriate mutations identified by DNA sequencing. The effect of the mutations on adherence by cotransformed bacteria will be quantified by three established adherence assays to type V collagen, human tracheal cells, and lung tissue sections. In subsequent experiments, type 3 fimbrial adhesin antibodies will be developed to assist in experiments to localize the adhesin on Klebsiella type 3 fimbrial filament as well as on mutant strains, developed in earlier experiments, that may demonstrate increased or decreased adherence. Additional experiments will utilize synthetic nonameric peptides, derived from the region(s) of the mrkD gene shown to be important, to determine the primary amino acid sequence required for adherence by the type 3 adhesin and to determine if these regions encompass epitopes recognized by adhesin-specific antisera. In the last group of experiments the applicant will use a novel method of allelic exchange to generate specific deletions in the Klebsiella mrk gene cluster within the native chromosomal DNA. Subsequently the impact on adherence by these specifically mutated Klebsiella will be assessed in the three adherence assays.

研究：呼吸道上皮附着被认为是一种 肺炎发展的早期阶段。产克雷伯氏菌 丝状细胞器，称为菌毛，携带黏附蛋白 称为粘附素的亚基。这项提案将进一步说明 克雷伯氏菌3型菌毛粘附素与 呼吸道上皮。初步实验将确定哪种DNA 3型菌毛粘附素基因mrkD的序列(S)对 粘附素的正常功能。两种必需的重组质粒 产生表达非粘附型3型菌毛的转化细菌 (PFK28)和3型菌毛粘附素蛋白(PFK58) 建造的。因此，在这项研究中，特定部位和随机突变 在pFK58中可以产生，并通过DNA鉴定适当的突变 测序。突变对共转化粘附性的影响 细菌将通过三种已建立的粘附性试验进行量化 V型胶原、人气管细胞和肺组织切片。在……里面 随后的实验中，3型菌毛粘附素抗体将 开发用于辅助在克雷伯氏菌上定位粘附素的实验 3型菌毛细丝和突变菌株，在 早期的实验，可能会显示增加或减少 坚持不懈。更多的实验将利用合成的非聚合体 来自mrkD基因区域(S)的肽，显示为 重要的是，要确定所需的初级氨基酸序列 通过3型粘附素的粘附性并确定这些区域是否 包括粘附素特异性抗血清识别的表位。在最后一次 一组实验申请者将使用一种新的等位基因方法 用于产生克雷伯氏菌mrk基因特异性缺失的交换 在天然染色体DNA中形成簇状。随之而来的影响 这些特定突变的克雷伯氏菌的依从性将在 三种依从性试验。