| Attaching and effacing (A/E) F. coli strains include human enteropathogens responsible for dehydrating infantile diarrhea, hemorrhagic colitis and the hemolytic uretic syndrome as well as for diarrheal disease in several animal species. All of these pathogens share highly-conserved virulence genes encoded on a 35kb pathogenicity island (PAT) termed the loccus of enterocyte effacement (LEE). The luxS gene and ler (LEE encoded regulator) gene are up-regulators of many of the virulence genes of the LEE, including eae gene which encodes an adherence intimin, an important outer membrane protein of the type III secretion system. For EHEC and EPEC, in vivo roles for luxS and ler in bacterial virulence have not been documented. The goals of this study were to genetically characterize luxS and ler of rabbit EPEC strains (REPEC 0103 and RDEC- 1) to determine the effects of deletion of luxS and ler on in vivo virulence. In order to determine whether deletion of the luxS or ler would attenuate the A/E strain while maintaining its immunogenicity, two kinds of internal deletion mutations in luxS and ler were respectively introduced into REPEC 0103 and RDEC-l using suicide delivery vector (pCVD442) technology. Although luxS deletion mutant of ROE-i showed no release of hormone-like molecule (an auto-inducer) , which increase in concentration as populations grow, it still partially maintained its virulence. The ler deletion mutants of REPEC 0103 and ROE-i showed increased responses in cell density and growth rates while completmentation of the entire ler gene restored the ability to normal levels. Rabbits were orogastrically inoculated with a single dose of the ler mutant of REPE 0103 and challenged with the virulent parent strain 2 weeks post immunization. Pre-inimune sera, and sera obtained 2 weeks post immunization, were used to determine IgG responses to whole bacteria. Following immunization and challenge, rabbits were observed daily for clinical signs of disease including weight change and diarrhea, and for fecal shedding of the inocula. At sacrifice, intestinal sections were obtained for quantitation of mucosally attaching bacteria. Post inoculation of the ler mutant of REPEC 0103, animals gained weight and experienced no diarrhea although they shed the organisms for up to 2 weeks. After immunization, animals all showed over3 times increased serum IgG titer to killed whole bacteria, but not to intimin. After wild-type challenge, immunized 53 rabbits exhibited normal weight gain, no diarrhea and, at sacrifice, had no mucosally adherent organisms. In contrast, unimmunized rabbits had severe diarrhea and weight loss and histological analysis revealed extensive mucosal colonization. The study demonstrates that a ler deletion mutation results in marked attenuation of REPEC 0103 virulence and maintains its irnmunogenicity. The immunization with the ler mutant of RDE-1 and challenge with RDEC- I HI 9A, which is a RDEC-1 derivative constructed by transformation of phage H19A carrying stx gene, showed that immune inoculation with the ler mutant provided all immunized rabbits with complete protection against RDEC-1 carrying phage H19A. The experiment also demonstrates that the ler deletion mutant of RDEC-l maintains its inimunogenicity while being sufficiently decreased in virulence. Therefore, both of the ler deletion mutants are safe attenuated vaccine candidates against rabbit enteropathogenic F. coli. This study provides the important information not only for human attenuated vaccine ag... |
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