Function Of MxiE Gene Of Shigella Flexneri 2a 2457T

Shigella spp.,commonly called Bacillus dysenteriae,are gram-negative,facultative anaerobes,non-spore forming pathogens.The pathogenesis of S.flexneri is based on the bacteria's ability to invade and replicate within the colonic epithelium,which results in severe inflammation and epithelial destruction.According to statistics of WHO,Shigellosis caused 1.1 million deaths and over 164 million cases each year,with the majority of cases occurring in the children of developing countries.The predominant serotypes of S.flexneri in developing countries were S. flexneri 2a.While in China,there were about 20 million cases each year,and S.flexneri 2a was responsible for 50-70%of those cases.Thus it is important to research on S.flexneri 2a for our sanitary and epidemic prevention.Expression and regulation of virulence gene was important for the invasion of Shigella. However,up to date little was known about relationship between Shigella's virulence gene and their pathogenesis.Thus,many researchers were focused on the study of ascertaining the function of those virulence factors and finding out unknown virulence substances.The mxi-spa locus on the virulence plasmid of S.flexneri encoded components of the typeⅢsecretion system,mxiE,a gene within this locus,encoded a protein that was homologous to the AraC/XylS family of transcriptional regulators.Currently its role in pathogenesis remained undefined.In order to clarify the function of mxiE in shigella,the deletion mutant S.flexneri 2a 2457T△mxiE::Kan was constructed with modifiedλ-Red recombination system.The growth curve of the mxiE mutant and the wild-type presented non-significant difference in LB medium. Compared with wild strain,the ability to utilize mannitol by mutants was higher,but lower in using glucose.The differential expression of virulent proteins was an important index to evaluate the gene function of mutants.In this study,the expression of virulent protein IpaB of the mutant mxiE and the wild-type strain was detected by Western Blotting.Moreover,2D-gel was used to identify the differential expression of the mutant and the wild-type strain at various growth phases induced by Congo red.It had been found that the mxiE mutant secreted IpaB(typeⅢeffectors required for host cell invasion) as the wild-type strain did.Potential targets for MxiE activation,PhoN2, OmpA and HtrA were down-regulated. Except for ascertaining the differential expression of virulent proteins,cell culturing(in vitro) and animal tests(in vivo) was also warranted,to evaluate the function of mxiE.In this study, eukaryotic cells e.g.HeLa cell line was used for invasion test of mxiE mutants.Sereny test with guinea pig and lung invasion test with Balb/c mouse were used,respectively,to verify the virulent of mild type and mutants.It had been demonstrated that the mxiE mutant retained the ability to invade mammalian cells in sereny test and tissue model,on the other hand,its evoking time and inflammation intensity was less efficient than that of wild-type.The mxiE mutant represented weakness virulence than the wild-type strain in vivo test,which indicated that other supplemented bypass was involved in its invasion.In conclusion,mxiE had little effects on the carbohydrate metabolism of Shigella flexneri 2a 2457T.Whereas deletion of mxiE resulted in the decrease of the invasion ability of Shigella flexneri.