FIMBRIAL ADHERENCE TO LUNG EPITHELIA BY KLEBSIELLA

研究：呼吸道上皮细胞的附着被认为是一种 肺炎发展的早期阶段。 克雷伯氏菌产生 携带粘附蛋白的丝状细胞器，称为菌毛 称为粘附素的亚单位。 这一建议将进一步突出 克雷伯菌3型菌毛粘附素负责粘附 呼吸上皮 最初的实验将确定哪些DNA 3型菌毛粘附素基因mrkD的序列对于 粘附素的正常功能。 两个必要的重组质粒 产生表达非粘附性3型菌毛的转化细菌 （pFK28）和3型菌毛粘附素蛋白（pFK58）之前被 构建了 因此，在这项研究中，位点特异性和随机突变 在pFK58中可以产生合适的突变，并通过DNA鉴定 测序 突变对共转化细胞粘附的影响 将通过三种已建立的粘附试验对细菌进行定量， V型胶原、人气管细胞和肺组织切片。 在 在随后的实验中，3型菌毛粘附素抗体将被 开发用于协助实验定位克雷伯氏菌上的粘附素 3型菌毛丝，以及突变株，发展中国家 早期的实验，可能证明增加或减少 坚持。 另外的实验将利用合成的九聚物 肽，来源于mrkD基因的区域， 重要的是，确定所需的一级氨基酸序列， 粘附3型粘附素，并确定这些区域是否 包括被粘附素特异性抗血清识别的表位。 在过去 一组实验中，申请人将使用一种新的方法， 交换以在克雷伯氏菌mrk基因中产生特异性缺失 在天然染色体DNA内的簇。 随后，影响 这些特异性突变的克雷伯氏菌的粘附性将在 这三种粘附性测定。

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.