Study On The Role Of YbtE Gene In Enteroaggregative Escherichia Coli

Abstract The term pathogenicity island (PAl) is a new concept in the field of Bacteriology. PAls, which may be associated with the emersion of new pathogens and the evolution of microbial virulence, have been found in many bacteria. As a member of PAls, high pathogenecity island (HPI) encoding an iron uptake system mediated by siderophore yersinabactin was first identified in pathogenic Yersinia and is greatly essential for the mouse virulence trait. Recently, it is known that I-IPI is also a part of the genome in certain diarrheagenic E. coli strains. But the structure and function of HPI in these strains is unclear. So this paper will focus on three parts below to discuss the HPI in diarrheagenic E. ccii. 1. The prevalence of HPI in 94 enterotoxigenic E. coil strains isolated from China Using PCR and DNA dot-blot hybridization assays, 94 enterotoxigenic E. coli(ETEC) strains isolated from patients with diarrhea in China were investigated to detect HPI genes. I 4%( 13/94) of the strains containing HPI in the asnT tRNA region. A deletion of 347bp was found in the integrase genes of three ETEC strains by sequence analysis. It is suggested that the HPI in ETEC may have at least two types: one harboring a intact integrase gene and the other a integrase gene with a deletion. 2.The cloning and sequence analysis of ybt U? ybtT and ybtE of the HPI in enteroaggregative E. ccii The ybt U ybIT and ybtE of the HPI in enteroaggregative E. coil (EAEC) 17-2 were cloned by PCR. These genes were also sequenced and compared with Yersinia corresponding genes. The ybtU ybtT and ybtE genes of strain 17-2 have more than 99% and 97% identity with those of Yps HPI and Yen HPI, respectively. The deduced amino acid sequences of these genes also has 99% and 97% identity with those of Yps HPI and Yen HPI, ?? respectively. All these demonstrate that the three important genes within the functional 揷ore?region of Yps HPI are highly conservative in EAEC. The clones and conclusions above shone lights on the construction of EAEC mutant and determining the function of these genes and the impact on EAEC virulence. 3. Study on the role of ybtE gene in enteroaggregative E. co/i ybtE from EAEC 17-2 was cloned and deleted with its internal around 1kb fragment and inserted by an selectable substitution with kan in vitro. The homologues exchange in vivo mediated by suicide vector pKTN7O 1 were induced spontaneously, which disrupted the ybtE on the chromosome of EAEA 17-2. This ybtE deletion mutant named EAEC-T offer us a very useful carrier for the study on the function of ybE. The experiments such as growth of EAEC-T under iron-deficient conditions, virulence testing in mouse, adherence to HEp-2 cell and biochemical reactions under iron-deficient conditions showed that ybtE has no significant effects on iron uptake, mouse virulence and aggregative adherence to HEp-2 cell. For the first time, we demonstrated the prevalence and structure characterization of HPI in ETEC strains isolated from China. The HPI is inserted into the asnT RNA locus and exists in more than two types. Also for the first time, we finished the construction of EAEC ybtE mutant and conducted a primary and exploratory study on the function of ybtE. For EAEC, ybtE has no significant effects on the iron uptake , aggregative adherence to HEp-2 cell and mouse virulence. These resul...