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

Impact of proteomics on anti-Mycobacterium tuberculosis (MTB) vaccine development.

Tuberculosis is a serious infection disease which causes more than two million deaths annually. The TB pandemic has continued despite widespread use of the only available licensed TB vaccine--Bacillus Calmette-Guerin (BCG). Additionally, the increasing incidences of multidrug resistant strains and coinfection with HIV mean that tuberculosis constitutes a growing global threat. Thus, improvement of the vaccination strategy against TB is an urgent need, requiring international cooperation of the research community. The completion of many mycobacterial genome sequences has greatly facilitated the global analysis at the transcriptome and proteome level. This in consequence has accelerated progress in the vaccinology field resulting in identification of a large numbers of antigens with potential in TB vaccines. This review concentrates on the proteomic contribution to TB vaccinology. At the end of the article some recent achievements of structural proteomics and developing an epitope-driven tuberculosis vaccine are presented

A Novel Insight into the Oxidoreductase Activity of Helicobacter pylori HP0231 Protein.

Altogether our results show that HP0231 is an oxidoreductase that catalyzes disulfide bond formation in the periplasm. We propose to call it HpDsbA

Bacterial strains and plasmids used in this study.

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

Redox state of HP0231 in WT, mutants: <i>dsbI</i>::<i>aph, hp0231</i>::<i>cat</i> and complemented strains.

<p>Bacterial cultures were treated with 10% TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) controls were separated by 18% SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0231 was performed. Each lane contains proteins isolated from the same amount of bacteria.</p