Thioloxidoreductase HP0231 of Helicobacter pylori impacts HopQ-dependent CagA translocation.

Thioloxidoreductase HP0231 of Helicobacter pylori plays essential roles in gastric colonization and related gastric pathology. Comparative proteomics and analysis of complexes between HP0231 and its protein substrates suggested that several Hop proteins are its targets. HP0231 is a dimeric oxidoreductase that functions in an oxidizing Dsb (disulfide bonds) pathway of H. pylori. H. pylori HopQ possesses six cysteine residues, which generate three consecutive disulfide bridges. Comparison of the redox state of HopQ in wild-type cells to that in hp0231-mutated cells clearly indicated that HopQ is a substrate of HP0231. HopQ binds CEACAM1, 3, 5 and 6 (carcinoembryonic antigen-related cell adhesion molecules). This interaction enables T4SS-mediated translocation of CagA into host cells and induces host signaling. Site directed mutagenesis of HopQ (changing cysteine residues into serine) and analysis of the functioning of HopQ variants showed that HP0231 influences the delivery of CagA into host cells, in part through its impact on HopQ redox state. Introduction of a C382S mutation into HopQ significantly affects its reaction with CEACAM receptors, which disturbs T4SS functioning and CagA delivery. An additional effect of HP0231 on other adhesins and their redox state, resulting in their functional impairment, cannot be excluded.

Impact of selected amino acids of HP0377 (Helicobacter pylori thiol oxidoreductase) on its functioning as a CcmG (cytochrome c maturation) protein and Dsb (disulfide bond) isomerase.

Helicobacter pylori HP0377 is a thiol oxidoreductase, a member of the CcmG family involved in cytochrome biogenesis, as previously shown by in vitro experiments. In this report, we document that HP0377 also acts in vivo in the cytochrome assembly process in Bacillus subtilis, where it complements the lack of ResA. However, unlike other characterized proteins in this family, HP0377 is a dithiol reductase and isomerase. We elucidated how the amino acid composition of its active site modulates its functionality. We demonstrated that cis-proline (P156) is involved in its interaction with the redox partner (CcdA), as a P156T HP0377 variant is inactive in vivo and is present in the oxidized form in B. subtilis. Furthermore, we showed that engineering the HP0377 active motif by changing CSYC motif into CSYS or SSYC, clearly diminishes two activities (reduction and isomerization) of the protein. Whereas HP0377CSYA is inactive in reduction as well as in isomerization, HP0377CSYS retains reductive activity. Also, replacement of F95 by Q decreases its ability to regenerate scRNase and does not influence the reductive activity of HP0377CSYS towards apocytochrome c. HP0377 is also distinguished from other CcmGs as it forms a 2:1 complex with apocytochrome c. Phylogenetic analyses showed that, although HP0377 is capable of complementing ResA in Bacillus subtilis, its thioredoxin domain has a different origin, presumably common to DsbC.

Bacterial thiol oxidoreductases - from basic research to new antibacterial strategies.

The recent, rapid increase in bacterial antimicrobial resistance has become a major public health concern. One approach to generate new classes of antibacterials is targeting virulence rather than the viability of bacteria. Proteins of the Dsb system, which play a key role in the virulence of many pathogenic microorganisms, represent potential new drug targets. The first part of the article presents current knowledge of how the Dsb system impacts function of various protein secretion systems that influence the virulence of many pathogenic bacteria. Next, the review describes methods used to study the structure, biochemistry, and microbiology of the Dsb proteins and shows how these experiments broaden our knowledge about their function. The lessons gained from basic research have led to a specific search for inhibitors blocking the Dsb networks.

Engineering of Helicobacter pylori Dimeric Oxidoreductase DsbK (HP0231).

The formation of disulfide bonds that are catalyzed by proteins of the Dsb (disulfide bond) family is crucial for the correct folding of many extracytoplasmic proteins. Thus, this formation plays an essential, pivotal role in the assembly of many virulence factors. The Helicobacter pylori disulfide bond-forming system is uncomplicated compared to the best-characterized Escherichia coli Dsb pathways. It possesses only two extracytoplasmic Dsb proteins named HP0377 and HP0231. As previously shown, HP0377 is a reductase involved in the process of cytochrome c maturation. Additionally, it also possesses disulfide isomerase activity. HP0231 was the first periplasmic dimeric oxidoreductase involved in disulfide generation to be described. Although HP0231 function is critical for oxidative protein folding, its structure resembles that of dimeric EcDsbG, which does not confer this activity. However, the HP0231 catalytic motifs (CXXC and the so-called cis-Pro loop) are identical to that of monomeric EcDsbA. To understand the functioning of HP0231, we decided to study the relations between its sequence, structure and activity through an extensive analysis of various HP0231 point mutants, using in vivo and in vitro strategies. Our work shows the crucial role of the cis-Pro loop, as changing valine to threonine in this motif completely abolishes the protein function in vivo. Functioning of HP0231 is conditioned by the combination of CXXC and the cis-Pro loop, as replacing the HP0231 CXXC motif by the motif from EcDsbG or EcDsbC results in bifunctional protein, at least in E. coli. We also showed that the dimerization domain of HP0231 ensures contact with its substrates. Moreover, the activity of this oxidase is independent on the structure of the catalytic domain. Finally, we showed that HP0231 chaperone activity is independent of its redox function.

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a ß-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231.

BACKGROUND: In the genome of H. pylori 26695, 149 proteins containing the CXXC motif characteristic of thioldisulfide oxidoreductases have been identified to date. However, only two of these proteins have a thioredoxin-like fold (i.e., HP0377 and HP0231) and are periplasm-located. We have previously shown that HP0231 is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. Although HP0377 was originally described as DsbC homologue, its resolved structure and location of the hp0377 gene in the genome indicate that it is a counterpart of CcmG/DsbE. RESULTS: The present work shows that HP0377 is present in H. pylori cells only in a reduced form and that absence of the main periplasmic oxidase HP0231 influences its redox state. Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin. However, it possesses disulfide isomerase activity, as it catalyzes the refolding of scrambled RNase. Additionally, although its standard redox potential is -176 mV, it is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, similar to E. coli DsbA or E. coli DsbC. The CcmG proteins that play a role in a cytochrome c-maturation, both in system I and system II, are kept in the reduced form by an integral membrane protein DsbD or its analogue, CcdA. In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD. Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori. CONCLUSIONS: The present data, in combination with the resolved three-dimensional structure of the HP0377, suggest that HP0377 is an unusual, multifunctional CcmG protein.

Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems.

The bacterial proteins of the Dsb family-important components of the post-translational protein modification system-catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp.

Identification and analysis of mutations in the katG gene in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

INTRODUCTION: A major role in the development of resistance of Mycobacterium tuberculosis to isoniazid (INH) is attributed to mutations in the katG gene coding for the catalase/peroxidase, an enzyme required for obtaining a pharmacologically active form of the drug. Analysis of mutations in the katG gene in M. tuberculosis strains may contribute to the development of reliable and rapid tests for detection of INH resistance. The aim of the study was to identify and characterize mutations in the katG gene in multidrug-resistant M. tuberculosis clinical isolates. MATERIAL AND METHODS: The study included 46 strains of M. tuberculosis, recovered from MDR-TB patients in Poland in 2004. Mutations in the katG gene were detected by comparing DNA sequences with the corresponding sequence of a wild-type reference laboratory strain (M. tuberculosis H37Rv). The obtained results were interpreted in the context of MIC values of INH and catalase activity of the strains tested. RESULTS: A total of 43 (93%) strains contained mutations in the katG gene. The most frequently observed were mutations at codon 315, found in 34 (74%) strains. Mutations at other codons were rare: 4 strains contained mutations at codon 463, 2 at codon 131 and another 2 at codon 234. Mutations at codons 68, 91, 101, 126, 128 and 194 were found in single strains only. Two strains, for which no mutations at codon 315 of the katG gene were identified, had a unique translation termination mutation, which would invariably result in polypeptide truncation leading to the generation of dysfunctional catalase polypeptides. Both these strains presented the highest MIC values for INH (80 and 100 µg/mL) and showed a complete loss of catalase activity. For the remaining 41 strains with katG mutations, the MICs of INH were within the range 0.2-10 µg/mL. Thirty-six (88%) of those strains retained their catalase activity. CONCLUSIONS: Mutations at codon 315 within the katG gene, depending on their type might be useful for the prediction of INH resistance. Whereas the missense mutations do not affect the catalase activity or the level of INH resistance, the nonsense mutations result in high-level resistance to INH and a total loss of catalase activity.