An Evolutionary Analysis of Fimbriae in Enterobacteria

肠杆菌菌毛的进化分析

• 批准号: 6783126
• 负责人: Daniel E. DYKHUIZEN
• 金额: $ 39.48万
• 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 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6783126
• 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）：该提案的主要重点是继续之前提案的工作，是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大肠杆菌的粘附素和其他基因对肠外感染，特别是尿路感染（UTI）的适应。主要尿路致病性血清型的克隆内遗传多样性将用于确定病理适应性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O 18：K1：H7血清型的菌株-一种主要的尿路致病性克隆。将确定编码各种粘附菌毛的基因簇内的核苷酸多态性，并用于表征克隆内的祖先/后代关系。然后，两个祖先和两个后代菌株将调查额外的突变变化，在多达一个兆碱基的基因组中使用新开发的技术，GIRAFF。在GIRAFF中，来自两个菌株的大小片段被熔化，再杂交并用错配特异性内切核酸酶CEL 1处理，该内切核酸酶能够以高特异性和灵敏度切割错配的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E.大肠杆菌基因组进行调查，大约有二十多个同义突变，预计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。将检测发现非同义突变的基因和发现突变的插入区域，以观察在其他6个尿路致病性克隆的相同DNA区域中是否发现类似突变。在这些其他克隆中的每一个中通常发现突变的那些区域将被认为在发病机制中是重要的。将研究这些突变的功能效应。这些致病适应性基因座的一个子集将在我们收集的125个大肠杆菌中测序。大肠杆菌菌株，其中包括致病性和致病性菌株，看看我们的新分析技术，区域分析，将确认这些变化是病理适应性的。如果是这样的话，这种方法可以用来发现致病适应性基因座从许多预期被测序的基因组的大肠杆菌。杆菌