| Cholera is an acute intestinal infection. As its rapidly spreading and the ability to cause large explosive outbreaks, cholera is one of the main reasons for diarrhea in the Asia and most of the Aferica. Since 1961, the world has been experiencing the seventh pandemic of cholera, and the causative pathogen of which is El Tor biotype of V. cholerae serogroup O1.As the pathogen which is responsible for the severe disease, Vibrio cholerae could be divided into over 200 serogroups based on the serotyping of O antigen. However,O1 and 0139 are the only ones that have been associated with epidemic disease.O1 could be divided into classical and El Tor in terms of biotyping, while 0139 is a kind of emergency infectious disease which was first reported in Southeast Asia in 1993.In V. cholerae, the cholera toxin (CT) is responsible for the symptoms, which is carried by the genome of a lysogenic bacteriophage called CTXΦ. In fact, CTXΦcould exist either as a prophage integradted into the chromosome or as a replicating plasmid, which means CTXΦcan be induced to be a kind of particles and propagate laterally from toxigenic strains to non-toxigenic strains, even causing the emergence of new epidemic strains. So, the study of CTXΦ-host interaction is very importment for understanding the evolution of epidemic V. cholerae.The early stage of CTXΦinfection is recognizing its receptor and the interaction between them. In this study, FITC conjugation, GST-fusion expression pull down technique and SPR-BIACORE were used to identify the direct interaction between TcpA and pⅢCTX.At first, TcpA and pⅢCTX of El Tor with his-tag were expressed and purified by affinity chromatography. The purified pⅢCTX was labeled with FITC and co-incubated with V. cholerae of El Tor (N16961) and classical (1119). The TcpA mutant strain 1119△TcpA was also constructed as a negative control. The result was observed under confocal microscopy. It proved that there was interaction between pⅢCTX of El Tor and V. cholerae of classical (1119), and the interactin was mediated by TcpA protein.Meanwhile, the ORF of N16961-pⅢCTX was cloned into plasmid pGEX-4T-1 and expressed in E. coli BL21 with GST-tag. Then, the purified protein was used to do the pull down assay, which proved that the pⅢCTX of El Tor could interact with TcpA from El Tor and classical biotyping directly. Furthermore, we used the surface Plasmon resonance biomolecular interaction analysis (SPR-BIACORE) to confirm the interaction between pⅢCTX and TcpA of El Tor in quantity. The purified pⅢCTX was coupled on the surface of the CM5 bissensor chip, while the TcpA solution was driven across the flowcell of the CM5 bissensor chip coupled the pⅢCTX protein, and the pⅢCTX protein could capture the TcpA protein if there were interaction between them. From the association and dissociation of the reaction process, the kinetic constant could be evaluated.In conclusion, we proved that there was direct interaction between pⅢCTX and TcpA in El Tor in this study. Also, we found that the interaction existed between N16961-pⅢCTX and O395-TcpA while they were negative between O395-pⅢCTX and N16961-TcpA, O395-pⅢCTX and O395-TcpA. These results indicated that the difference of interaction between pⅢCTX and TcpA from different serogroups may be responsible for their epidemic or not in the history of cholera, and further study will be done to prove our suggestions. |
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