Biochemical Characterization Of Disulfide Oxidoreductase DsbA And Its Function In Haemophilus Parasuis

Haemophilus parasuis is the causative agent of Gl?sser’s disease and colonises in pigs. It produces fibrinous polyserositis, arthritis and meningitis causing severe systemic disease. Disulfide bond oxidoreductase Dsb A catalyzes disulfide formation in proteins to form three dimension structure which underpins proteins function. Bacteria encode multiple Dsb As, but the type and number of Dsb A homologues varies enomously from one species to another. Two Dsb A homologues were found in H. parasuis. One of them shows 47% protein identities to Dsb A of Escherichia coli K-12, while the other has no significant protein similarities to Ec Dsb A. Their functions haven’t been reported in any articles yet. It is ensential for us to find out the importance of Dsb As in H. parasuis.This study mainly focused on the Dsb As’ biochemical characterization and phenotypes to understand the roles Dsb As play in H. parasuis by constructing dsb A1 and dsb A2 gene deletion mutants of SH0165. Details are as follows.1. Construction of dsb A deletion mutants and its complement strainTo gain the mutants, we first cloned the upstream and downstream homology arms of dsb A1 and dsb A2 from SH0165 genome separately and amplified kan or erm resistance cassettes from p SHK3 or p AT18. After that, overlapping arms with the right resistance cassettes were used to construct p K18-dsb A1-UKD, p K18-dsb A2-UED, p SHK3-dsb A1-UKD and p SHK3-dsb A2-UED recombinant vectors. The mutants were successfully obtained by natural-transforming vectors into SH0165. Next, we cloned dsb A1 coding sequence with its promoter and terminator to shutter vector p SHK3,the recombinant was methylation modificatied in vitro and transformed into Δdsb A1 to obtain the complement strain.2. Biochemical characterization analysis of Dsb A1 and Dsb A2 underpins their enyme functionFirst of all, we demonstrated that the dsb A gene deletion mutants and the wild strain SH0165 cause a same sensitivity to the redox-active metal copper in copper-oxidation assay(0~10m M). We proposed Dsb A1 and Dsb A2 couldn’t refold the misfolded proteins caused by copper which means they may not exbibit disulfide isomerase activity in vivo.Second, prokaryotic expression vector of Dsb A1, Dsb A2 and Trx A was constructed to gain the purified proteins. We investigated the Dsb As’ disulfide reductase activity in a specific assay, using insulin as a substrate. Three of them catalyzed the reduce of insulin, but Dsb As catalyzed the reaction less efficiently than Trx A with a lag phase of about 20 min in the presence of the reductant DTT. Both Dsb A1 and Dsb A2 were proved not to be an isomerase in vitro.Last but not least, dsb A mutants are detected by the ability to grow under 1m M DTT. The survival rate of strains survived in a concentration of 1m M DTT was measured. We found that SH0165 and Δdsb A2 showed a similar sensitivity to DTT, while Δdsb A1 seemed more sensitive comparing to them, implying that Dsb A1 exhibit a disulfide oxidase to balance the DTT reduction in vivo.3. dsb A1 is involved in the growth of SH0165In order to analyze the role of dsbA1 in H. parasuis, growth curves of dsb A1 mutant and the wild-type SH0165 strain were conducted providing that the deletion of dsb A1 resulted in great reduce in both OD600 and the amount of bacteria. The complement strain restored the growth phenotype. In other words, compared to the wild-type SH0165 strain, the deletion of dsb A1 led to a significant growth defect.To determine at what step the growth process of the mutant was blocked, we observed the bacterial forms when they grew to 6h, 12 h or 24 h respectively by scanning electron microscopy(SEM). Part of the mutant’s forms changed to be longer in morphology obviously when grew to 6h and 12 h while recovered to 24 h as compared with the parental strain SH0165. Therefore, the growth defect appeared due to a defect in bacterial dividing process.Iron-chelated growth assay was tested to illustrate the growth restriction. The tested strain Δdsb A1 and the parental strain SH0165 grew on TSA/V/S with or without 250μM and 300μM Dip. The result showed that the mutant Δdsb A1 grew poorly as compared with SH0165 in iron chelated assay. It suggested that Dsb A1 played an important role in iron uptake.4. Other functions of dsb A1Our research put emphasis on serum resistance to explore the role dsbA1 played in H. parasuis. It turned out that Δdsb A1 showed great defect compared with the wild type after interacting with 90% pig serum.