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

DsbA. PLoS ONE 9(9): e106247. doi:10.1371/journal.pone.0106247 Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA Anna D. Grabowska 0 Ewa Wywia 0 Stanislaw Dunin-Horkawicz 0 Anna M. asica 0 Marc M. S. M. Wo sten 0 Anna Nagy-Staron 0 Renata Godlewska 0 Katarzyna Bocian-Ostrzycka 0 Katarzyna Pien kowska 0 Pawe aniewski 0 Janusz M. Bujnicki 0 Jos P. M. van Putten 0 E. Katarzyna Jagusztyn-Krynicka 0 Luis Eduardo Soares Netto, Instituto de Biociencias - Universidade de Sao Paulo, Brazil 0 1 Department of Bacterial Genetics, Institute of Microbiology, University of Warsaw , Warsaw , Poland , 2 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland, 3 Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, the Netherlands, 4 World Health Organization Collaborating Centre for Reference and Research on Campylobacter/ World Organisation for Animal Health Reference Laboratory for Campylobacteriosis , Utrecht , The Netherlands , 5 Institute of Science and Technology , Klosterneuburg , Austria , 6 Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University , Poznan , Poland 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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Additional data are available upon request. Funding: This work was funded by the grant of Polish Ministry of Science and Higher Education (grant No. N401 183 31/3968 and N N303 550 439). EW has been supported by the Polish Ministry of Science and Higher Education (grant POIG.02.03.00-00-003/09). SDH has been supported by the National Science Centre (NCN, grant 2011/03/D/NZ8/03011) and by the Polish Ministry of Science and Higher Education (MNiSW, fellowship for outstanding young scientists). JMB has been supported by the 7th Framework Programme of the EU (grant HEALTHPROT, contract number 229676) and by the statutory funds of IIMCB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Bacterial proteins of the Dsb (disulfide bond) system catalyze the formation of disulfide bridges, a post-translational modification of extra-cytoplasmic (periplasm-located, membrane-anchored or secreted) proteins, which leads to stabilization of their tertiary and quaternary structures and often influences activity of their protein substrates. In Gram-negative bacteria, the process of oxidative folding takes place in the periplasm, whereas in Grampositive bacteria it occurs in the space between the cytoplasmic membrane and the cell wall [1,2]. The Dsb system has been studied in detail in Escherichia coli K-12 (EcDsb), where it operates in two antagonistic, partially coinciding metabolic pathways, based on the oxidation and the reduction/isomerization reactions [3,4,5,6,7]. The first reaction (catalyzed by EcDsbA and EcDsbB) appears as the non-selective formation of disulfide bonds in newly synthesized proteins [8], whereas the second (driven by EcDsbC and EcDsbD) ensures the rearrangement of improperly introduced disulfides. Given the importance of disulfide bond formation to achieve native protein structures, the number of crystallographic studies of DsbA-homologous proteins has risen sharply in the last decade, as reflected by the structures deposited in the Protein Data Bank (PDB) for thirteen of non-redundant, functionally characterized DsbA homologs, ten from Gram-negative and three from Grampositive bacteria [9]. Despite a common thioredoxin (TRX) fold, members of the DsbA superfamily display numerous structural differences, which result in their various redox properties and substrate specificities, as reviewed by McMahon et al. [9]. The delineated differences include, for instance, the sequence of the XX dipeptide within the active-site CXXC motif, which is present in the form of a CPHC in EcDsbA and more than 70% of its homologs [10,11,12]. The diverse redox properties of the DsbAs, as well as other TRX-fold proteins, are assumed to be also determined by a residue preceding the CXXC motif and by a residue upstream of the cis-Proline loop [13,14], as well as by indirect interactions of polar residues with the side chain of the N-terminal catalytic cysteine residue [15]. Previous reports [16,17,18,19] and our recent updated examination (Figure 1) have revealed that the systems of disulfide bond formation in bacteria are extremely diverse, often involving multiple Dsb homologs and functional analogs. In E. coli K-12 two monocistronic units, dsbA and dsbB, which encode the m (...truncated)