Preliminary Study Of Effectors NleB1and NleB2from Escherichia Coli O157:H7

Enterohemorrhagic E. coli(EHEC)O157:H7are non-invasive and gram-negative pathogens which are detrimental to human health because people who infected with O157:H7will suffer from diarrhea, haemorrhagic colitis and haemolytic uraemic syndrome (HUS). The attachment to the surface of intestinal epithelial cells is the key to the development of disease. Various virulence effectors can be injected into host cells by type Ⅲ secretion system(TTSS), and these proteins collaboratively subvert host physiological function by formation of actin-rich ’pedestal’ and disruption of cell-cell junction. O157:H7not only induces an’Attaching and Effacing’(A/E) lesion on the surface of intestinal epithelial cells, but escapes from the recognition of the pathogen by the host’s innate immune system.Virulence associated protein type Ⅲ secretion system (T3SS) is a highly conserved’molecular nanosyringes’secretion machinery, consisting of more than20proteins. The deletion of escR gene, which encodes T3SS structural protein EscR, will induce T3SS function deficient mutant of O157:H7and affect the attachment to host cells.NleB1and NleB2are38kDa proteins conserved across A/E pathogens, while C. rodentium carries only a single copy of the gene. The NleB family of effectors are found on two pathogenicity islands (IE6and PP4) encoding genes for nleB1and nleB2respectively. NleB from C. rodentium and NleB1from EPEC can both inhibit the TNF-induced NF-κB activation.The gene z4328encoding hypothetical protein NleB1(Z4328) has89%identities with nleB of C. rodentium, while z0985encoding hypothetical protein NleB2(Z0985) has only62%identities using blasting analysis. Both of these hypothetical effectors have conserved catalytic DxD motif, which is also present in NleB. It is speculated that NleB1(Z4328), a homolog of NleB, is likely to have similar biochemical mechanism. To find whether NleB1and NleB2are effectors that can inject into host cells by T3SS will lay the foundation for the follow-up studies. The recombinant DNA fragments, obtained kanamycin resistance gene as well as upstream and downstream gene of escR, were constructed by overlap extension PCR, and then transferred into EDL933/pKD46to replace escR gene by λRed homologous recombination. The T3SS-deficient mutant (△escR) was successfully construted. Compared with the wild-type EDL933, the growth rate and attachment on HeLa cells of△escR were significantly decreased. The deletion of escR gene, which encodes T3SS structural protein EscR, affects the attachment of EDL933, suggesting better basis for future studies of T3SS.The prokaryotic expression vector pGEX-2TK-NleB2was constructed and induced with IPTG to obtain soluble protein GST-NleB2. Use recombinant protein with high purity to immune the mice and produce a polyclonal antibody to GST-NleB2. The expression of NleB2from wild-type O157:H7can be detected by the polyclonal antibody.We cloned gene z0985and z4328amplified from the EDL933genome to prokaryotic expression vector pFLAG-MAC, and then respectively transfered the recombinational plasimds into wide-type EDL933strain and T3SS-deficient mutant (△escR). The expression level of Flag-NleB1and Flag-NleB2proteins in different strains was identified by Western blot. The recombinational proteins was only detected in filtered culture medium of WT strains, suggesting T3SS-deficient mutant (△escR) cannot secret Flag-NleB1and Flag-NleB2into host cells. HeLa cells were infected with different strains containing transferred plasimds or remained uninfected. Cells were washed, extracted and subjected to western blot analysis with anti-flag antibodies. Flag-NleBl and Flag-NleB2was found be localized in cytoplasm of HeLa cells infected with wild-typed strains, but we did not detect any proteins in cells infected with T3SS-deficient mutants. This suggests that Flag-NleB1and Flag-NleB2need the help of T3SS to be translocated into host cells.In this study, we constructed the T3SS deficient mutant of O157:H7EDL933, and illuminated the escR deletion mutant strain affects the attachment of EDL933. NleB1and NleB2proteins are effectors that can be injected into host cells by T3SS. We also obtained anti-GST-NleB2polyclonal antibody, as well as eukaryotic and prokaryotic vectors which can express NleB1or NleB2with different tags. Our works laid a basis for future studies of interactions between O157:H7and host cells, and further studies are still needed to understand the virulence and pathogenic mechanisms of O157:H7.