Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

<p>Abstract</p> <p>Background</p> <p>Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in <it>Campylobacter jejuni </it>is more complex than the one in the laboratory <it>E. coli </it>K-12 strain.</p> <p>Results</p> <p>In the <it>C. jejuni </it>81-176 genome, the <it>dsb </it>genes of the oxidative pathway are arranged in three transcriptional units: <it>dsbA2</it>-<it>dsbB</it>-<it>astA, dsbA1 </it>and <it>dba</it>-<it>dsbI</it>. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in <it>dsb </it>gene regulation was proven by a reporter gene study in a <it>C. jejuni </it>wild type strain and its isogenic <it>fur </it>mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind <it>in vitro </it>to the <it>C. jejuni </it>promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives <it>dsbA1 </it>gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary <it>dba-dsbI </it>mRNA structure for <it>dsbI </it>mRNA translation was verified by estimating individual <it>dsbI </it>gene expression from its own promoter.</p> <p>Conclusions</p> <p>The present work shows that iron concentration is a significant factor in <it>dsb </it>gene transcription. These results support the concept that iron concentration - also through its influence on <it>dsb </it>gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The <it>dba </it>expression is not only essential for the translation of the downstream <it>dsbI </it>gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI.</p

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

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<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

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

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