Functional Study Of DsbC In Vibrio Cholerae

Cholera is an acute diarrhoeal infection caused by the bacterium Vibrio Cholera,which provokes disease exclusively in human.Some patients present with profuse watery diarrhea and vomiting.Bile salts enhance the activity of the transmembrane transcription activator Tcp P by inducing intermolecular disulfide bonds in its periplasmic domain.Disulfide bonds are important for the correct folding of many secreted proteins in both prokaryotes and eukaryotes.In these proteins,disulfide bonds stabilize protein structure and protect the thiol group from over-oxidation.In Gramnegative bacteria,it is therefore critical that the correct disulfide bonds are formed in the proteins that have muitiple free thiol.The pathways of disulfide bond formation in Gram-negative bacteria have been best characterized in Escherichia coli.Vc Dsb C and Ec Dsb C were purified by nickel ions resin and make polyclonal antibody.To differentiate whether TC directly affects Vc Dsb C or Ec Dsb C,we investigated the interaction between TC and the proteins by using isothermal titration calorimetry(ITC),TC could bound Vc DsbC and Ec Dsb C.To investigate the reductase activity of Vc DsbC and if the direct interaction of TC and Vc Dsb C alters Vc Dsb C activity,we tested Vc Dsb C for its ability to catalyze the reduction of insulin in the presence or absence of TC.Under the standard assay conditions,VcDsb C catalyzed insulin reduction by dithiothreitol(DTT)and had higher reductase activity and TC had no effect on Vc Dsb C reductase activity.We engineered an additional non-consecutive disulphide into the ampicillin-resistance protein ?-lactamase(a “PDI detector”)and suggested that Vc Dsb C had isomerase activity in vivo.In this study,we investigated the reductase activity and isomerase activity of Vc Dsb C.We also constructed a method that can detect isomerase activity in vitro.This method could help us investigate Vc Dsb C further.