Construction And Characterization Of EHEC 0157 Ler/stx Gene Deletion Mutant

Enterohemorrhagic Escherichia coli (EHEC) are important human and animal pathogens which are capable of causing bloody and nonbloody diarrhea, hemorrhagic colitis (HC) and potentially fatal hemolytic uremic syndrome (HUS). The most common serotype of EHEC is O157:H7. One of the important virulence trait of EHEC O157:H7 is the capacity to produce attaching and effacing (A/E) lesions on enterocyte. This property encoded by a 43kb pathogenicity island (PAI) termed the locus of enterocyte effacement (LEE). The genes of LEE are organized into four polycistronic operons, LEE1, LEE2, LEE3 and LEE4. LEE1 contains ler (LEE-encoded regulator) gene (previously known as orfl). ler is the first open reading frame of LEE. The product of ler transcriptionally activates LEE2, LEES and LEE4. The ler gene product is a central up-regulator of many of the virulence genes of the LEE.Another important virulence factor of EHEC O157:H7 was Shiga-toxin(Stx), which was encoded by a bacteriophage inserted into the chromosome of O157:H7. This potent cytotoxin is the factor that leads to death and many other symptoms in patients infected with EHEC.In this study, in order to obtain an attenuated vaccine candidate, a ler/stx deletion mutant of O157:H7 was constructed. The fragment of internal 279bp deletion mutation of ler was amplified by PCR, and cloned into a suicide vector pCVD442, which containing the ler-dependent R6K replicon and sacB gene. The recombinant plasmid pCVD442:: &ler was transformed into a host cell SMIOp/r. pCVD442::A/er was then transfered from SM10(pCVD442:: Afer) into EHEC O157:H7 EDL933 by conjugation, with selection for nalidixic acid and ampicillin resistance. Resulting conjugants were plated onto LB agar (supplemented with 5% sucrose, but without NaCl) and incubated overnight at 29 癈. Sucrose-resistant and ampicillin-sensitive colonies were selected and screened by PCR. In the resultant, ler gene of chromosome exchanged with the deletion ler gene of suicide vector by homologous recombination, and EHEC O157:H7 ler deletion mutant was constructed.Due to potential instability of the phage carrying the stx gene, stx (or the phage) was lost with serial passages of bacteria in LB. And it was confirmed by PCR and53sequencing. A lerlstx deletion mutant of EHEC O157:H7 was constructed. The supernatant of lerlstx deletion mutant was inoculated into vero cells, which indicating that O157 lerlstx deletion mutant lost the toxigenicity to vero cells.Group I and group II of mice were orogstrically inoculated respectively with the lerlstx deletion mutant and the virulent parent strain. Mice were observed daily for clinical signs of disease including weight change. After inoculation of the deletion mutant, group I of mice gained weight normally and experienced no clinical signs. In contrast, group II of mice exhibited weight loss and all dead in four days. This study demonstrates that O157 lerlstx deletion mutant might be an attenuated vaccine candidate.