Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast

AIM: Bacillary dysentery caused by Shigella flexneriis still a threat to human health. Of four invasion plasmid antigen proteins (IpaA, B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. METHODS: By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKr-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AHL09. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for β-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid. RESULTS: Among the 2x106 transformants, 64 positive clones were obtained as determined by activation of His,Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM. CONCLUSION: These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S.flexneri infection.

AIM:Bacillary dysentery caused by Shigella flexneri is still a threat to human health.Of four invasion plasmid antigen proteins (IpaA,B,C and D),IpaC plays an important role in the pathogenicity of this pathogen.The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. METHODS:By applying two-hybrid system,the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC.The bait plasmid was transformed AH109,and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AH109.The transformation mixture was plated on medium lacking Trp,Leu,and His in the initial screen,then restreaked on medium lacking Trp,Leu,His and Ade.Colonies growing on the selection medium were further assayed for β- galactosidase activity.BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid. RESULTS:Among the 2×10~6 transformants,64 positive clones were obtained as determined by activation of His, Ade and LacZ reporter genes.Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein,RanBPM. CONCLUSION:These results provide evidence that IpaC may be involved in the invasion process of S.flexneri by interacting with RanBPM,and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S. flexneri infection.