Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA.

BACKGROUND: Bacterial 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 RESULTS: Phylogenetic 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. CONCLUSIONS: Our 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

Harde demonstraties versus een gastvrij welkom voor vluchtelingen: Een casestudy over burgerparticipatie omtrent de noodopvang van vluchtelingen en de effecten hiervan op de houding van de burgers.

<p>The diagrams illustrate mean values and standard deviations of AstA activity derived from three experiments; for each experiment the AstA activity were carried out in triplicate. Statistical significance was calculated using Student <i>t</i> test for comparison of independent groups (GraphPad Prism) with reference to the AstA activity in the wild type (WT) strain. P values of P<0.05 were considered statistically significant (*).</p

Oligonucleotides used in the present study.

<p><b>Bold letters</b> indicate <i>C. jejuni</i> nucleotide sequences; restriction recognition sites introduced for cloning purposes are <u>underlined</u>, complementary fragments of primers Cj872up and Cj872dw are marked with <i>italics</i>. Most primers were based on the <i>C. jejuni</i> 81116 nucleotide sequence, but for some experiments, previously designed primers based on the <i>C. jejuni</i> NCTC or 81–176 nucleotide sequences were used, or primers were designed to introduce point mutations. Their single pair mismatches with <i>C. jejuni</i> 81116 are <u>double underlined</u>.</p><p>Oligonucleotides used in the present study.</p

Homology models of <i>C. jejuni</i> DsbA1 and DsbA2.

<p><i>C. jejuni</i> DsbA1 and DsbA2 (CjDsbA1 and CjDsbA2) models built on <i>E. coli</i> DsbA [EcDsbA (PDB ID: 2ZUP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106247#pone.0106247-Inaba1" target="_blank">[80]</a>)] and DsbL [EcDsbL (PDB ID: 3C7M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106247#pone.0106247-Grimshaw1" target="_blank">[22]</a>)], experimentally characterized members of the DsbA superfamily. Structural representations are shown in ribbon view (A, D, G and J). Electrostatic surfaces coloured by charge from red, acidic, -1kT to blue, basic, +1kT. The orientation in B, E, H and K follows the orientation in the top row (A, D, G and J) and in C, F, I and L is rotated by 130 degrees around the vertical axis, clockwise.</p

Taxonomic distribution of the DsbA DsbB/I and AstA in gamma-Proteobacteria and epsilon-Proteobacteria.

<p>Color boxes indicate the presence of the classical DsbA and DsbB (two inner rings), DsbA1-2, DsbB2, AstA, and DsbI (four outer rings) in a given genome.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Autoagglutination of <i>C. jejuni</i> 81116 strains: wild type (WT), <i>cjdsbA1^-</i>, <i>cjdsbA2^-</i>, <i>cjdsbB^-</i> and <i>cjdsbI^-</i> mutants.

<p>Bacterial autoagglutination was monitored as a decrement of turbidity (A) or optical density (B) of bacterial suspension in LB at room temperature after harvesting cells from BA plates. The <i>cjdsbA1^-</i> strain does not autoagglutinate, contrary to the wild type (WT), <i>cjdsbA2^-</i>, <i>cjdsbB^-</i> and <i>cjdsbI^-</i> strains. The figure presents a representative result.</p

Alkaline phosphatase PhoX activity in <i>C. jejuni</i> 81116 strains: wild type (WT), <i>cjdsbA1^-</i>, <i>cjdsbA2^-, cjdsbB^-</i> and <i>cjdsbI^-</i> mutants.

<p>The diagrams illustrate mean values and standard deviations of PhoX activity derived from three experiments; for each experiment the PhoX activity were carried out in triplicate. Statistical significance was calculated using Student t test for comparison of independent groups (GraphPad Prism) with reference to the PhoX activity in the wild type (WT) strain. P values of P<0.05 were considered statistically significant (*).</p

Redox state of CjDsbA1 in <i>C. jejuni</i> 81116 strains: wild type (WT), <i>cjdsbA2^-</i>, <i>cjdsbB^-</i> and <i>cjdsbI^-</i> mutants.

<p>Bacterial cultures were treated with 10% TCA, followed by alkylation with AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid). Cellular proteins including the reduced (red; DTT treated, modified by AMS) controls were separated by 15% SDS-PAGE under non-reducing conditions, followed by Western blot analysis using antibodies against CjDsbA1. Each lane contains proteins isolated from the same amount of bacteria. The relative positions of protein molecular weight standard (MP) are listed on the left (in kilodaltons). The figure presents a representative result.</p

Insulin reduction assay for CjDsbA1 and CjDsbA2.

<p>The reaction mixture contained 150 µM insulin in potassium phosphate buffer, pH 7.0 and 2 mM EDTA. The assay was performed in the absence (▪) or presence of 10 µM EcDsbA (♦), CjDsbA1 (⁃) and CjDsbA2 (▴). Reactions started by adding DTT to the final concentration of 0.33 mM and the changes in the absorbance at 650 nm as a function of time were measured. The figure presents a representative result.</p