| Virulence genes of pathogenic bacteria can be located on the chromosome where they may be organized as so-called pathogenicity island (PAI). PAI is not only attributed to the pathogenicity of bacteria and specific phases of the infection progress but also play an important role in the evolution of bacterium. Many PAI have been discovered in many pathogens to humans (Pathogenic E.coli,Yersiniae spp.,Salmonella spp.,Vibrio cholerae et al.)- Among them,the PAI of Yersiniae which encodes siderophore yersiniabactin (Ybt) system is designated High-pathogenicity island (HPI). HPI is essential for the mouse virulence in Yersiniae. The core part of HPI comprises genes for the biosynthetic,regulatory and transport of Ybt. Recently,the HPI appears to be widespread in various enterobactia strains,for example,in disrrheagenic E.coli strains,Citrobacter diversus,and various species of Klebsiella and non-I serotypes of Salmonella enterica. Now,the research on Yersinia HPI in the enterobactia is reported mostly from the viewpoint of molecular epidemiology,but the structure and function of HPI in these strains is unclear.Enteroaggregative E.coli (EAggEC),since the first described in 1987,has been classified the fifth disrrheagenic E.coli. EAggEC is defined with its adherence to Hep-2 cells in aggregative pattern. The mechanisms of its pathogenesis are not entirely clear. Schubert et al reported that HPI was found in 93% EAggEC,27% enteroinvasive E.coli,5% enteropathogenic E.coli and enterotoxigenic E.coli. The reason that different distribution frequency of HPI in various E.coli is unclear,either. As the high frequency distribution of HPI in EAggEC,this study will focus on the following parts to discuss the function of Yersiniae High-pathogenicity island in enteroaggregative E.coli.1. The prevalence of HPI and functional expression of Yersiniabactin inEAggEC strains isolated from ChinaUsing PCR methods,two EAggEC type strains,EAggEC 17-2 and EAggEC O42,and 6 EAggEC strains isolated from patients with diarrhea in China were investigated to detect HPI genes. The results show that there are 7 strains HPI positive except 1 strain HPI negative. HPI in 6 strains of 7 HPI-positive strains is inserted into asnT-tRNA site.The expression of fyuA gene,which encoded FyuA,the receptor of Ybt,was upregulated by extracellular Ybt level. Supernatants of EAggEC grown under normal condition (LB medium) and iron deficiency condition (LED medium) were applied to the reporter strain,WA-CS irpl::KN(pCJG3.3N),which was transferred the reporter plasmid pCJG3.3N contained promoter region of fyuA gene fused to gfp. Flow cytometry measurements were done to detect the changing of fluorescent signal of the reporter strain and give expression situation to Ybt indirectly. 7 EAggEC HPI-positive strains revealed an enhanced fluorescence signal but 1 EAggEC HPI-negative did not so. However,there is difference in expression condition and expression degree of Ybt among these 7 EAggEC HPI-positive strains. In addition,we detect that UFT073 strain lose the functional expression of Ybt although carrying the complete HPI core part. The results suggested that EAggEC HPI-positive strains expressed the Ybt system,but the presence of HPI core part doesn't mean Ybt expression.2. Research on the function of ybtP,ybtQ and ybtX genes in EAggEC 17-2ybtP,ybtQ and ybtX genes from Yen WA were cloned respectively and mutated,then inserted by a selectable substitution with chloramphenicoKcaf) gene in vitro. The fragment containing mutant gene was subcloned into suicide vector pKNG101,named pKH6,pKH 7 and pKH8 respectively. Moreover,EAggEC 17-2 was used to construct gene mutants by homologous recombination. Using conjunction mobilization,we screened the mutant of EAggEC 17-2 with the mutant phenotype by one step.Compared mutant and EAggEC 17-2,the experiments such as growthunder iron-deficient conditions,virulence testing to the mouse,and Ybt biosynthesis and regulation showed that ybtP,ybtQ and ybtX genes have no significant effects on iron uptak...
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