An Evolutionary Analysis of Fimbriae in Enterobacteria

描述（由申请人提供）：本提案的主要重点是设计出确定哪些突变参与了细菌作为人类病原体的适应性进化的方法，该提案继续了先前提案的工作。这里使用的模型系统是大肠杆菌粘附素和其他基因对肠道外感染，特别是尿路感染（UTI）的适应。主要尿路致病血清型的克隆内遗传多样性将用于确定致病突变靶向的基因位点，即当生物体是病原体时在环境中选择的基因变化。我们将详细研究属于O18:K1:H7血清型的菌株-一种主要的尿源性克隆。编码各种黏附膜的基因簇内的核苷酸多态性将被确定并用于表征克隆内的祖先/后代关系。然后，将使用新开发的技术GIRAFF，对两个祖先和两个后代菌株进行最多1兆碱基基因组的额外突变变化调查。在GIRAFF中，来自两个菌株的大小片段被熔化，重新杂交，并用错配特异性内切酶CEL 1处理，该酶能够以高特异性和敏感性切割错配DNA区域。然后使用多kb DNA探针进行Southern blot杂交鉴定CEL i特异性条带。在被调查的20%的大肠杆菌基因组中，预计将有大约24个同义突变，并将用于确定克隆的日期。所有其他突变将作为潜在的病理适应性变化进行分析。将对发现非同义突变的基因和发现突变的中间区域进行测试，以查看是否在其他六个尿源性克隆的相同DNA区域中发现了类似的突变。在这些其他克隆中通常发现的突变区域将被认为在发病机制中起重要作用。这些突变的功能效应将被研究。我们将在收集的125株大肠杆菌菌株（包括共生菌株和致病性菌株）中对这些致病性基因座的一个子集进行测序，看看我们的新分析技术，区域分析，是否会证实这些变化是致病性的。如果是这样的话，这种方法可以用于从大肠杆菌的许多预期测序基因组中发现致病适应性位点。

DESCRIPTION (provided by applicant): The major emphasis of this proposal, which continues the work of the previous proposal, is to devise ways to determine which mutations are involved in adaptive evolution of bacteria as human pathogens. The model system used here is the adaptation of adhesins and other genes of Escherichia coli to extra intestinal infections, particularly urinary tract infections (UTI). The within-clonal genetic diversity of the major uropathogenic serotypes will be used to determine gene loci targeted by pathoadaptive mutations, i.e. gene changes that are selected in the environment when the organism is a pathogen. We will study in detail strains belonging to O18:K1 :H7 serotype - a major uropathogenic clone. Nucleotide polymorphisms within the gene clusters encoding various adhesive fimbria will be determined and used to characterize ancestral/descendent relationships within the clone. Then, two ancestral and two descendant strains will be surveyed for additional mutational changes in up to one megabase of genome by using the newly developed technique, GIRAFF. In GIRAFF, sized fragments from two strains are melted, rehybridized and treated with the mismatch-specific endonuclease, CEL 1, that is capable of cutting mismatched DNA regions with high specificity and sensitivity. The CEL I-specific bands are then identified by Southern blot hybridization using multi-kb DNA probes. Within the 20% of the E. coli genome to be surveyed, about two-dozen synonymous mutations are expected and will be used to date the clone. All other mutations will be analyzed as potential pathoadaptative changes. The genes in which the nonsynonymous mutations are found and the intervening regions where mutations are found will be tested to see if similar mutations are found in the same regions of DNA in six other uropathogenic clones. Those regions commonly found with mutations within each of these other clones will be assumed to important in pathogenesis. The functional effects of these mutations will be investigated. A subset of these pathoadaptive loci will be sequenced in our collection of 125 E. coli strains, which includes both commensal and pathogenic strains, to see if our new analytic technique, zonal analysis, will confirm that these changes are pathoadaptive. If so, this approach can be used to discover pathoadaptive loci from the many expected to be sequenced genomes of E. coli.