Analysis Of Type Three Secretion System Chaperone Protein Complexes Of EHEC O157:H7by Tandem Affinity Purification

Enterohemorrhagic Escherichia coli (EHEC) O157:H7is an important causive agent of human intesinal disease which expresses a Type Three Secretion System (TTSS) playing a significant role in colonisation and responsible for its pathogenesis for human and animal host. TTSS is composed of structural proteins, secreted tranlocator proteins, secreted effector proteins and chaperones, all of which contributed to the virulence of EHEC. In the present study, a complexomics approach composed of tandem affinity purification (TAP) and blue native PAGE (BN PAGE) is developed. Subsequently, separation and purification of protein complexes containing target virulence protein (CesT or CesD) and identification of novel virulence proteins were conducted with the developed approach.The experimental scheme of this study can be detailed as followed:Firstly, TAP-tagged virulence proteins were expressed in O157:H7and bacteria were then mildly lysed for the preparation of protein complexes sample. Secondly, complexes containing TAP-tagged virulence proteins were purified and enriched by tandem affinity purification and separated by BN PAGE and then protein components were identified by LC-MS/MS. At last, the interactions between identified protein components of the complexes were analyzed and comfirmed by GST pull-down.Two vectors, pNTAP and pCTAP were designed and constructed, both expressing target proteins, with TAP tag in N-terminus and C-terminus respectively. Then TAP and BN PAGE technologies were successfully established based on the preliminary experiment. Complexes containing target protein GroEL were subsequently purified and identified by employing the established technologies without non-specific complexe contamination, demonstrating high specificity of the constructed technologies. Type Three Secretion System chaperone protein CesT and CesD were then fused with TAP tags to purify protein complexes. Further analysis showed complexes containing target protein without non-specific contamination. EF-Tu and GK (glycerol kinase), as protein components of complex containing CesT, were analyzed by GST pull-down and confirmed being able to interact directly with CesT in vivo.Results of this study demonstrated:Highly efficient complexomics technologies have been developed, which could purify protein complexes containg tagged target protein from E.coli with high specificity and analyze protein interaction between components of complex. These techonologies could be used in not only E.coli, but also other pathogens like shigella. This study provided fundamental theoretical and experimental bases for further studies, such as constructing protein intercation network of TTSS relevant virulence proteins and overall studies of mechanisms of TTSS.