Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

Abstract

BackgroundBacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.Methods and ResultsPhylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.ConclusionsOur results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.

Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment

“…S1). Single deletion mutants in dsbA and dsbB and a double dsbAB mutant were constructed and it was confirmed, as reported by Grabowska et al (2014), that these mutants (and the dsbD strain) showed a significant reduction in activity of the known Dsb-dependent enzyme alkaline phosphatase ( Fig. S1A).…”

“…Single deletion mutants in dsbA and dsbB and a double dsbAB mutant were constructed and it was confirmed, as reported by Grabowska et al . (), that these mutants (and the dsbD strain) showed a significant reduction in activity of the known Dsb‐dependent enzyme alkaline phosphatase (http://onlinelibrary.wiley.com/doi/10.1111/mmi.13008/suppinfo). However, haem blots of periplasmic fractions showed no major differences in dsbA , dsbB or dsbAB mutants compared with the wild type (http://onlinelibrary.wiley.com/doi/10.1111/mmi.13008/suppinfo).…”

“…The involvement of two other key dsb genes, dsbA ( cj0872 or dsbA1 according to Grabowska et al ., ) and dsbB ( cj0865 ), in cytochrome c biogenesis was also determined (http://onlinelibrary.wiley.com/doi/10.1111/mmi.13008/suppinfo). Single deletion mutants in dsbA and dsbB and a double dsbAB mutant were constructed and it was confirmed, as reported by Grabowska et al .…”

“…However, we found in this study that deletion of dsbA (cj0872) or dsbB had no major effect on cytochrome c biogenesis in C. jejuni NCTC 11168, which would argue against this model. The C. jejuni Dsb system is complex, with some strains encoding additional homologues of DsbA and DsbB (Grabowska et al, 2014). Nevertheless, it would be interesting to overexpress dsbA in C. jejuni and see if this has a similar effect to cccA deletion.…”

“…The involvement of two other key dsb genes, dsbA (cj0872 or dsbA1 according to Grabowska et al, 2014) The positions of CccA, CccB and CccC (identified by their mass and from haem blots of the respective deletion mutants) are indicated by arrows. B. Dithionite-reduced minus air-oxidised difference spectra of periplasmic fractions (1 mg ml −1 total protein) of wild type (black spectrum), dsbD mutant (red spectrum) and cj1207c mutant (blue spectrum).…”