An Evolutionary Analysis of Fimbriae in Enterobacteria

肠杆菌菌毛的进化分析

• 批准号: 6864886
• 负责人: Daniel E. DYKHUIZEN
• 金额: $ 37.76万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6864886
• 关键词: Escherichia coli; adhesin; alleles; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial genetics; biochemical evolution; endonuclease; evolution; gene expression; gene mutation; genetic strain; laboratory mouse; microorganism culture; nucleic acid probes; pathologic process; pilus; polymerase chain reaction; single strand conformation polymorphism; southern blotting; urinary tract infection; virulence

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.

描述(由申请人提供)：这项提案的主要重点是继续前一项提案的工作，旨在设计方法来确定哪些突变参与细菌作为人类病原体的适应性进化。这里使用的模型系统是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。主要致病血清型的克隆内遗传多样性将被用来确定致病适应突变所针对的基因位点，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O18：K1：H7血清型的菌株-一种主要的泌尿系致病克隆。编码各种粘附性菌毛的基因簇内的核苷酸多态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调查两个祖先和两个后代菌株在多达一兆数据库的基因组中的额外突变变化。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，CEL 1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探针通过Southern印迹杂交鉴定CEL I特异性条带。在要调查的20%的大肠杆菌基因组中，预计会有大约24个同义突变，并将用于确定克隆的日期。所有其他突变都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，以确定在其他六个泌尿系致病克隆的DNA相同区域中是否发现了类似的突变。通常在这些克隆中发现突变的那些区域将被认为在发病机制中具有重要作用。这些突变的功能影响将被调查。这些病理适应基因座的子集将在我们收集的125株大肠杆菌中进行测序，其中包括共生菌株和致病菌株，以查看我们的新分析技术--区带分析--是否将确认这些变化是病理适应的。如果是这样的话，这种方法可以用来从许多预计将被测序的大肠杆菌基因组中发现致病适应基因座。