Giardia , Entamoeba , and Trichomonas Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases)

Abstract

Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity

“…Although the precise mechanistic reasons for this counterintuitive observation remain to be determined, 5-NIs can be activated by different microbial pathways and probably target multiple microbial molecules (11,12,23) whose relative importance may depend on the compound and microbe. Because the specific target molecules of 5-NI drugs are not fully understood (23), a strategy focused on achieving effective whole-cell antimicrobial activity in the relevant systems may be the best choice for making rapid progress in developing improved antimicrobials in the clinically indispensable 5-NI class.…”

“…Mz resistance is functionally heterogeneous, with several causative mechanisms for each of the target microbes and between different microbes (7,24). For example, resistance in Giardia involves down-regulation of nitro drug-activating systems, including different reductases and redox proteins and metabolites (11,25), whereas Trichomonas also produces resistance proteins that directly detoxify nitro drugs (12) and Bacteroides has transporters that can remove nitro drugs from the cell (26). Despite this mechanistic diversity, for each of the resistant microbes, we were able to identify nitro compounds that could combat resistance, and some compounds were broadly effective against different resistant microbes.…”

“…In some cases, Mz resistance can be overcome by treatment with other 5-NI drugs, but many resistant microbial strains exhibit cross-resistance between the major currently available 5-NI drugs (9). Multiple mechanisms have been implicated in 5-NI drug resistance, including a diminished capacity to reduce and activate 5-nitro prodrugs (10,11) and detoxification of nitro drug radicals (12).…”

Emerging Metronidazole Resistance in Anaerobes and Mapping Their Susceptibility Behaviour

“…The nitroreductase enzyme assay was carried out according to the method described by Pal et al (2009). The reaction mixture comprised of Tris-acetate (100 mM Tris-HCl, 50 mM acetate buffer, pH 7.0), 30 µM metronidazole as a substrate, half MIC of different drugs as inhibitors, 0.3 mM NADPH or NADH and enzyme in a 1 mL reaction volume.…”

Screening for antibacterial and antiprotozoal activities of crude extracts derived from Mexican medicinal plants

“…On the other hand, trichomoniasis, a sexually transmitted disease associated with vaginitis, cervicitis, urethritis, prostatitis, epididymitis, cervical cancer, and infertility, accounts for 180 million infections acquired annually worldwide (Ali and Nosaki, 2007;Nanda et al, 2006). E. histolytica, T. vaginalis, and G. lamblia infections are mainly treated with imidazole type drugs, but it has been reported that these three protist pathogens have developed drug resistance (Upcroft and Upcroft, 2001;Pal et al, 2009). This has produced an urgent need for more efficient and safer antiprotozoal agents.…”