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

Roszczenko P, Grzeszczuk M, Kobierecka P, Wywial E, Urbanowicz P, Wincek P, Nowak E, Jagusztyn-Krynicka EK - BMC Microbiol. (2015)

Bottom Line: Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. paula.roszczenko@gmail.com.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Redox state of HP0377 in wt and mutant: hp0265::aph measured for cells grown in the absence (a) or presence (b) of DTT. 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 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
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Fig8: Redox state of HP0377 in wt and mutant: hp0265::aph measured for cells grown in the absence (a) or presence (b) of DTT. 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 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria

Mentions: Dsb proteins that play a role in a cytochrome-c biogenesis, in both system I and system II, are kept in the reduced form by the integral membrane protein DsbD, or its shortened analogue CcdA. Both proteins catalyze the transfer of electrons from cytoplasmic thioredoxin across the inner membrane to the periplasm; however the mechanism of the process in the case of CcdA is still unknown [48]. Apart from HP0265, which was described as CcdA, a search for Dsb homologs in the H. pylori genome, employing the E. coli Dsb sequence as a query sequence, revealed that H. pylori contains another DsbD homolog (i.e., HP0861). Similar to Rodobacter capsulatus CcdA and Bacillus subtilis CcdA, HP0265 is a six-transmembrane (TM) protein containing two cysteines at the end of the first and the fourth TM segments [48]. HP0861, however, has five cysteines with an unusual, and as yet uncharacterized, spatial arrangement, and it could be involved in a process other than the cytochrome c biogenesis. Thus, to establish whether HP0265 is responsible for re-reducing HP0377, we created a H. pylori strain lacking hp0265. The recombinant plasmid pUWM2019 (based on a vector non-replicating in Helicobacter cells) was used for the mutagenesis. The plasmid contains the hp0265 gene disrupted by insertion of a kanamycin resistance cassette into the gene coding sequence. The hp0265 mutants were only produced in the presence of the reducing agent DTT, which indicates the importance of HP0265 and its reducing activity for bacterial survival. We then tested whether deletion of hp0265 affects the redox state of HP0377. We noticed that growth of the hp0265 mutated strain without DTT was much slower than growth of the wt strain (Fig. 7). Therefore, to check the influence of HP0265 on the HP0377 redox state, the H. pylori N6 hp0265− strain was cultivated 24 h longer than the wt strain to achieve the same cell density. As shown in Fig. 8a, the knock-out of hp0265 results in the accumulation of a noticeable amount of HP0377 in the oxidized state, which indicates the role of HP0265 in re-reducing HP0377. The addition of DTT to the medium resulted in the presence of HP0377 in the reduced form, clearly verifying the role of HP0265 (Fig. 8b).Fig. 7


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

Roszczenko P, Grzeszczuk M, Kobierecka P, Wywial E, Urbanowicz P, Wincek P, Nowak E, Jagusztyn-Krynicka EK - BMC Microbiol. (2015)

Redox state of HP0377 in wt and mutant: hp0265::aph measured for cells grown in the absence (a) or presence (b) of DTT. 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 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491210&req=5

Fig8: Redox state of HP0377 in wt and mutant: hp0265::aph measured for cells grown in the absence (a) or presence (b) of DTT. 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 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
Mentions: Dsb proteins that play a role in a cytochrome-c biogenesis, in both system I and system II, are kept in the reduced form by the integral membrane protein DsbD, or its shortened analogue CcdA. Both proteins catalyze the transfer of electrons from cytoplasmic thioredoxin across the inner membrane to the periplasm; however the mechanism of the process in the case of CcdA is still unknown [48]. Apart from HP0265, which was described as CcdA, a search for Dsb homologs in the H. pylori genome, employing the E. coli Dsb sequence as a query sequence, revealed that H. pylori contains another DsbD homolog (i.e., HP0861). Similar to Rodobacter capsulatus CcdA and Bacillus subtilis CcdA, HP0265 is a six-transmembrane (TM) protein containing two cysteines at the end of the first and the fourth TM segments [48]. HP0861, however, has five cysteines with an unusual, and as yet uncharacterized, spatial arrangement, and it could be involved in a process other than the cytochrome c biogenesis. Thus, to establish whether HP0265 is responsible for re-reducing HP0377, we created a H. pylori strain lacking hp0265. The recombinant plasmid pUWM2019 (based on a vector non-replicating in Helicobacter cells) was used for the mutagenesis. The plasmid contains the hp0265 gene disrupted by insertion of a kanamycin resistance cassette into the gene coding sequence. The hp0265 mutants were only produced in the presence of the reducing agent DTT, which indicates the importance of HP0265 and its reducing activity for bacterial survival. We then tested whether deletion of hp0265 affects the redox state of HP0377. We noticed that growth of the hp0265 mutated strain without DTT was much slower than growth of the wt strain (Fig. 7). Therefore, to check the influence of HP0265 on the HP0377 redox state, the H. pylori N6 hp0265− strain was cultivated 24 h longer than the wt strain to achieve the same cell density. As shown in Fig. 8a, the knock-out of hp0265 results in the accumulation of a noticeable amount of HP0377 in the oxidized state, which indicates the role of HP0265 in re-reducing HP0377. The addition of DTT to the medium resulted in the presence of HP0377 in the reduced form, clearly verifying the role of HP0265 (Fig. 8b).Fig. 7

Bottom Line: Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. paula.roszczenko@gmail.com.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus