| ETEC (Enterotoxigenic Escherichia coli) is the principal pathogenic bacteria leading to diarrhea of human and young animals (newborn lambs, calves and weaned piglets), and its high morbidity and mortality of post-weaning diarrhea cause great economic loss in the pork industry. K88ac+ETEC is the predominant pathogen of domestic post-weaning diarrhea, of which the major virulence factors are adhesins, enterotoxin, endotoxin, edema disease toxin, hemolysin, etc. Virulence factors of ETEC, including adhesive fimbriae, heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), have drawn great attention over the recent years. However, rare research focused on hemolysin which serves as key virulence factor of Uropathogenic Escherichia coli (UPEC) and Enterohemorrhage Escherichia coli (EHEC). Therefore, this study targets at hemolysin of K88ac+ETEC to explore the role of hemolysin in pathogenicity of ETEC. In order to investigate the biological functions and the pathogenic mechanism of HlyA encoded by hlyA gene of Enterotoxigenic Escherichia coli, the anti-HlyA polyclonal antibody from rabbits was obtained in this study. A pair of the primers based on the sequence of ETEC strain C83902 in GenBank were designed to amplify the hlyCA gene by PCR, and the product was inserted into the multiple clone sites of pET-28a(+) vector. The recombinant vector pET-28a-hlyC4 was confirmed by the combined restriction enzyme digestion and DNA sequencing. Then the E.coli BL21(DE3) with transformed recombinant plasmid were induced with IPTG to express the HlyA protein. After purification by Ni2+-chelating chromatography, the recombinant protein was used to immunize the New Zealand rabbits in two ways to obtain polyclonal antibody with high specificity. The anti-HlyA polyclonal antibody was applied in the study of the biological functions of HlyA, which can lay the foundation for further research of HlyA of Enterotoxigenic Escherichia coli in the pathogenesis.Based on the hlyA deletion mutant and its complementary strain which were conducted from ETEC strain C83902, we investigated the interaction between hemolysin and other virulent factors of ETEC through realtime PCR test, as well as the existence forms of hemolysin expressed in vitro, and we also designed a serial of experiments on porcine jejunum epithelial cells IPEC-J2, including bacterial adherence and inhibition assay, observation of cellular morphology and cytotoxicity test to explore role of hemolysin in pathogenic mechanism of ETEC. Results of Real-time PCR showed that level of sepA transcripts in △ hlyA mutant was 83% more than in WT (p<0.01), which might indicate certain coadjustment and interaction between HlyA and SepA. On the contrary, deletion of hlyA down-regulated fimH and eltB by 40% and 21% respectively (p<0.01), as well as faeG by 24%(p< 0.05). The relationship between hemolysin and these virulence factors associated with ability of adherence suggested probability that hemolysin was related to process of adherence to host cells. Besides, there was no significant difference of fliC transcripts between △ hlyA mutant and WT. Moreover, we isolated outer membrane vesicles (OMV) and hemolysin to identify the existence of free HlyA as well as OMV-associated HlyA in the bacterial culture supernatant, which comfirmed the hypothesis that hemolysin can employ OMV as vehicle to transport to host cells. In addition, bacterial adherence assays of IPEC-J2 cells revealed that adherence ability of △ hlyA mutant increased 2.13-fold of WT (p<0.01). And we also found morphological changes of the IPEC-J2 cells exposed to hemolysin, such as more round shape and smaller size. Additionally cytotoxicity test demonstrated the ability of HlyA to induce cell death. In conclusion, we speculated that both cytolytic activity of HlyA and degradation of host protein triggered by HlyA could lead to exfoliation of epithelial cells which facilitated the discharge of bacteria. And this supposition coincided with results of bacterial adherence assays. |
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