Potential Involvement of Several Nitroreductases in Metronidazole Resistance in Helicobacter pylori

Jorgensen, Margaret; Trend, Mark A.; Hazell, Stuart L.; Mendz, George L.

doi:10.1006/abbi.2001.2427

Cited by 24 publications

(23 citation statements)

References 50 publications

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“…3A) in the presence of plumbagin, given that the total proteins were loaded in comparable amounts (OmpC was used as an internal control; data not shown). Gene mdaB is known as "modulator of drug activity B" and has been recently proposed to function as an NADPH quinone reductase inducible by menadione (15,19). Our results further demonstrated that mdaB also responded to plumbagin.…”

Section: Resultssupporting

confidence: 76%

Expression Analysis of Up-Regulated Genes Responding to Plumbagin inEscherichia coli

et al. 2006

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Plumbagin is found in many medicinal plants and has been reported to have antimicrobial activities. We examined the molecular responses of Escherichia coli to plumbagin by using a proteomic approach to search for bacterial genes up-regulated by the drug. The protein profile obtained was compared with that of E. coli without the plumbagin treatment. Subsequent analyses of the induced proteins by mass spectroscopy identified several up-regulated genes, including ygfZ, whose function has not been defined. Analyses of the 5-flanking sequences indicate that most of these genes contain a marbox-like stretch, and several of them are categorized as members of the mar/sox regulon. Representatives of these genes were cloned into plasmids, and the marbox-like sequences were modified by site-directed mutagenesis. It was proven that mutations in these regions substantially repressed the level of proteins encoded by the downstream genes. Furthermore, plumbagin's early effect was demonstrated to robustly induce SoxS rather than MarA, an observation distinctly different from that seen with sodium salicylate.Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a naphthoquinone having antibacterial (6), antifungal (5), anticancer (22), and antimutagenic activities (7). Like other redoxcycling chemicals such as paraquat and menadione, plumbagin has been used as an agent to generate superoxide or reactive oxygen species in order to study oxidative stress (14). Plumbagin has been suggested to activate SoxS by oxidizing the SoxR molecule (10, 21) or inhibiting the repression of MarR (2), the effect of which is exerted on marA, resulting in activation of the mar/sox regulon. In addition, it has been observed that the expression of some members of the mar/sox regulon in Escherichia coli, such as sodA (8), nfo (3), ribA (20), and pqi (21), is up-regulated by treatment with plumbagin.Although plumbagin could induce excessive expression of superoxide dismutase and catalase, overexpression of sodA failed to protect E. coli (17). The toxic effect of plumbagin may not simply result from the production of reactive oxygen species. It has been reported that plumbagin inhibits NADH dehydrogenase, as well as causing respiratory arrest (17). Plumbagin has also been shown to modify the lactose carrier and inhibit its binding with galactoside; the modified carrier then becomes completely inactive (29). The above effects appeared more or less to result directly from the chemical nature of plumbagin. In this report, we focus on the responses of the bacteria to the chemical, in which multiple proteins were simultaneously induced by plumbagin treatment. We report the evaluation of the bacterial regulatory systems after treating E. coli with plumbagin. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. E. coli JM109 was used as the major experimental strain for the chemical treatments and the cloning host. Plasmids pGEM-T-easy (Promega, Madison, WI), pBluescript II SKϩ (Stratagene, La Jolla, CA), and pQE60 (QIAGEN, Valencia, CA) wer...

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Section: Resultssupporting

confidence: 76%

Expression Analysis of Up-Regulated Genes Responding to Plumbagin inEscherichia coli

et al. 2006

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“…This result is in agreement with Marais et al 33 who suggested that an MTZ resistance phenotype may arise in H. pylori without mutation in rdxA or frxA. Interestingly, several groups 22,[34][35][36][37] and genome sequence annotation [38][39][40] suggest other putative redox systems that may play a role in MTZ resistance. These include fdxB (encoding a ferredoxin-like protein), fdxA (ferredoxin), fldA (flavodoxin), oorD (the g-subunit of 2-oxoglutarate oxidoreductase and porD (the g-subunit of pyruvate ferredoxin oxidoreductase).…”

Section: R5 G C T a C -A A A A A T T C T A A A A A A A T A A A G G A supporting

confidence: 88%

Genetic analysis of Helicobacter pylori clinical isolates suggests resistance to metronidazole can occur without the loss of functional rdxA

Kim¹,

Joo²,

Lee³

et al. 2009

J Antibiot

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Resistance to metronidazole (MTZ) in Helicobacter pylori is associated with mutations in rdxA, encoding an oxygen-insensitive NADPH nitroreductase, and mutations in frxA, encoding a NAD(P)H-flavin oxidoreductase. Despite this association, the strict correlation of MTZ resistance with mutations in rdxA or frxA is still controversial. In this study, rdxA allelic replacement was used to distinguish resistance-associated nucleotide mutations from the natural genetic diversity of H. pylori. Replacement with truncated rdxA resulted in MTZ resistance, whereas replacement with missense-mutated rdxA from resistant clinical isolates failed to yield MTZ resistance. Thus, although truncation of rdxA confers MTZ resistance in G27 H. pylori, MTZ resistance found in other clinical isolates is not due to the identified amino-acid substitutions. Three of our MTZ-resistant clinical isolates expressed functional rdxA and two of these also encoded full-length frxA. Therefore, MTZ resistance can arise in H. pylori possessing functional rdxA, suggesting that other factors are involved in MTZ resistance.

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“…The genome of H. pylori encodes a variety of redox-active enzymes, many of which were identified through functional assays or through mutation-based strategies (2,9,15,25,30,31,42). Since a large percentage of genes in H. pylori are essential for viability (6), it is likely that some genes associated with redox activities, such as fqrB (HP1164), would not be identified in genetic screens.…”

Section: Discussionmentioning

confidence: 99%

Flavodoxin:Quinone Reductase (FqrB): a Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter pylori and Campylobacter jejuni

Maurice¹,

Cremades

Croxen

et al. 2007

J Bacteriol

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Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k cat of 122 s ؊1 , and an apparent K m of 14 M for menadione and 26 M for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate-and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO 2 , and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO 2 fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO 2 -requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO 2 fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.

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Potential Involvement of Several Nitroreductases in Metronidazole Resistance in Helicobacter pylori

Cited by 24 publications

References 50 publications

Expression Analysis of Up-Regulated Genes Responding to Plumbagin inEscherichia coli

Expression Analysis of Up-Regulated Genes Responding to Plumbagin inEscherichia coli

Genetic analysis of Helicobacter pylori clinical isolates suggests resistance to metronidazole can occur without the loss of functional rdxA

Flavodoxin:Quinone Reductase (FqrB): a Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter pylori and Campylobacter jejuni

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