Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

Müller, Joachim; Samuel, Rout; Leitsch, David; Vaithilingam, Jathana; Hehl, Adrian B.; Müller, Norbert

doi:10.1016/j.ijpddr.2015.03.001

Cited by 45 publications

(58 citation statements)

References 45 publications

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“…Indeed, DNA damage may be sufficient to arrest the cell cycle, as suggested by transcriptional downregulation of the mitotic regulator Mad2 under Mtz and previous reports of H2A phosphorylation after sublethal Mtz exposure (15,70,71). Second, H 2 O 2 requires only bivalent reduction to form H 2 O, but the Mtz prodrug requires reduction with as many as six electrons until it forms an inert amine, making it more reactive (10,72). This requirement for intracellular enzymatic reduction may, however, impose limits on the rate of Mtz activation which do not apply to H 2 O 2 .…”

Section: Discussionmentioning

confidence: 89%

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis

Ansell

McConville

Baker

et al. 2016

Antimicrob Agents Chemother

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dUnderstanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and ␣-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control. Giardia duodenalis (synonyms, Giardia lamblia and Giardia intestinalis) is a parasitic protist of the vertebrate gastrointestinal tract and the most common parasite of humans (1). The vegetative life cycle stage (trophozoite) infects around 1 billion people and causes 200 to 300 million cases of diarrheal disease (giardiasis) each year (1). Infection with G. duodenalis is also associated with the development of chronic diseases such as irritable bowel syndrome, chronic fatigue, and diabetes (2). At present, only two major drug classes are available to treat giardiasis: nitroimidazoles, primarily metronidazole (Mtz), and benzimidazoles, such as albendazole (3,4). Resistance to Mtz is documented in treatment-resistant clinical isolates (5-8), and resistant lines can be generated in vitro (reviewed in reference 9).The mechanism of action of Mtz is relatively poorly understood. This compound is thought to diffuse into G. duodenalis trophozoites as an inactive prodrug, whereupon it is enzymatically reduced (activated), yielding reactive intermediates. Mtz intermediates are thought to kill trophozoites by oxidizing proteins, lipids, and DNA (10, 11); however, the relative importance of damage to different biomolecules for Mtz-induced cytotoxicity is contested (10, 12). Resistance to Mtz involves changes in enzyme expression which limit drug activation. Nitroreductase-1, thioredoxin reductase, and pyruvat...

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Section: Discussionmentioning

confidence: 89%

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis

Ansell

McConville

Baker

et al. 2016

Antimicrob Agents Chemother

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“…Recombinant nitroreductase-1 reduces Mtz when NADH is supplied as an electron donor (Müller et al, 2015). This NADH-dependent mechanism is reminiscent of canonical nitroreductases that reduce Mtz at the NADH-binding site (Koder and Miller, 1998; Martinez-Julvez et al, 2012), and may therefore be catalytically independent of the ferredoxin domain.…”

Section: Discussionmentioning

confidence: 99%

Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines

et al. 2017

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Giardia duodenalis is an intestinal parasite that causes 200–300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID10, 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid α-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID10 diverged from those in 713-r and WB-r (r ≤ 0.2), which were more similar to each other (r = 0.47). In 106-2ID10, a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

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“…Indeed, gOCT has been co-precipitated with GlNR2, a nitroreductase known to reduce MTZ and nitazoxanide (Müller et al., 2015). Similarly, GASP-180 interacts with the other Giardia nitroreductase GlNR1, also active toward several nitrocompounds (Müller et al., 2015). The direct involvement of GlNR1 and GlNR2 on NBDHEX nitroreduction to form intermediates reactive with gOCT and gGASP-180 could be taken into account.…”

Section: Discussionmentioning

confidence: 99%

Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis

Camerini

Bocedi

Cecchetti

et al. 2017

International Journal for Parasitology: Drugs and Drug Resistan

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Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-γ (gEF1Bγ), and structural proteins, e.g. α-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1Bγ and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis.

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Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

Abstract: HighlightsG. lamblia has two nitroreductases with substrate specificities not only for nitro compounds, but also for quinones.GlNR1 rather activates nitro drugs by forming toxic intermediates, GlNR2 rather inactivates them.

Cited by 45 publications

References 45 publications

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis

Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines

Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis

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