C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning

Abstract

Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.

Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis

“…Next, we tested the ability of the CjDsbAs to introduce disulfide bonds in vitro using ribonuclease A (RNaseA) as its substrate; RNaseA needs four properly located disulfide bonds for its enzyme activity. The oxidative activity of the DsbAs was assessed by spectrophotometrically measuring the RNaseA cleavage of cCMP (cyclic-2′,3′cytidinemonophosphate) (Figure 1) [35,36]. We found that both CjDsbAs recognized RNaseA as their substrate and exhibited oxidase activity such as that of EcDsbA, in contrast to EcDsbL that, according to literature data, does not introduce disulfides into RNaseA [24].…”

Genotypic and Phenotypic Characterization of blaNDM–7-Harboring IncX3 Plasmid in a ST11 Klebsiella pneumoniae Isolated From a Pediatric Patient in China

“…Disulfide bond formation is a crucial step in the folding process of a protein and is catalyzed by bacterial proteins of the Dsb system. DsbC is responsible for rearranging incorrect disulfides introduced between cysteine residues ( Banaś et al, 2020 ).…”

Harnessing the potential of bacterial oxidative folding to aid protein production

“…These proteins vary widely. They can share as little as 7.37% identity with E. coli K‐12 DsbA (Banaś et al., 2020), sometimes, like DsbC, they form oligomers (Qin et al., 2009), and they can have either broad or narrow substrate specificities (Lin et al., 2009), all the while maintaining oxidase function. Moreover, fusions of DsbA with other proteins or protein domains often occur in prokaryotes.…”