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1.  Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia 
Objectives
The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia.
Methods
PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTRr) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance.
Results
We demonstrated that several lines of highly MTRr G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTRr Giardia. However, reduction of flavins is suppressed in highly MTRr cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTRr Trichomonas vaginalis.
Conclusions
These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.
doi:10.1093/jac/dkr192
PMCID: PMC3133484  PMID: 21602576
metronidazole; ronidazole; tinidazole; Blastocystis; NADPH oxidase
2.  Impaired Parasite Attachment as Fitness Cost of Metronidazole Resistance in Giardia lamblia▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4643-4651.
Infections with the diarrheagenic protozoan pathogen Giardia lamblia are most commonly treated with metronidazole (Mz). Treatment failures with Mz occur in 10 to 20% of cases and Mz resistance develops in the laboratory, yet clinically, Mz-resistant (Mzr) G. lamblia has rarely been isolated from patients. To understand why clinical Mzr isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mzr cell lines with stable, high-level resistance to Mz and significant cross-resistance to tinidazole, nitazoxanide, and furazolidone. Oral infection of suckling mice revealed that three of five Mzr cell lines could not establish infection, while two Mzr cell lines infected pups, albeit with reduced efficiencies. Failure to colonize resulted from a diminished capacity of the parasite to attach to the intestinal mucosa in vivo and to epithelial cells and plastic surfaces in vitro. The attachment defect was related to impaired glucose metabolism, since the noninfectious Mzr lines consumed less glucose, and glucose promoted ATP-independent parasite attachment in the parental lines. Thus, resistance of Giardia to Mz is accompanied by a glucose metabolism-related attachment defect that can interfere with colonization of the host. Because glucose-metabolizing pathways are important for activation of the prodrug Mz, it follows that a fitness trade-off exists between diminished Mz activation and reduced infectivity, which may explain the observed paucity of clinical Mzr isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.
doi:10.1128/AAC.00384-11
PMCID: PMC3186953  PMID: 21825286
3.  A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro 
The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardia lamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID90 values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 μM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID90 values around 100 μM (MTR-susceptible isolates typically have an ID90 of 5–12.8 μM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells.
doi:10.1016/j.ijantimicag.2010.03.004
PMCID: PMC3103471  PMID: 20456926
Pyruvate:ferredoxin oxidoreductase; Tinidazole; Ronidazole; 5-Nitroimidazole; Cross-resistance
4.  Synthesis and Electrochemistry of 2-Ethenyl and 2-Ethanyl Derivatives of 5-Nitroimidazole and Antimicrobial Activity against Giardia lamblia 
Journal of medicinal chemistry  2009;52(13):4038-4053.
Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole (5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl and 2-ethanyl 5-NI derivatives, we found that compounds with a saturated bridge between the 5-NI core and a pendant ring system exhibited only modestly increased antigiardial activity and could not overcome Mz resistance. By contrast, olefins with a conjugated bridge connecting the core and a substituted phenyl or heterocyclic ring showed greatly increased antigiardial activity without toxicity, and several overcame Mz resistance and were more effective than Mz in a murine giardiasis model. Determination of the half-wave potential of the initial one-electron transfer by cyclic voltammetry revealed that easier redox activation correlated with greater antigiardial activity and capacity to overcome Mz resistance. These studies show the potential of combining systematic synthetic approaches with biological and electrochemical evaluations in developing improved 5-NI drugs.
doi:10.1021/jm900356n
PMCID: PMC2766634  PMID: 19480409
5.  5-Nitroimidazole Drugs Effective against Metronidazole-Resistant Trichomonas vaginalis and Giardia duodenalis 
Metronidazole (Mz)-resistant Giardia and Trichomonas were inhibited by 1 of 30 new 5-nitroimidazole drugs. Another five drugs were effective against some but not all of the Mz-resistant parasites. This study provides the incentive for the continued design of 5-nitroimidazole drugs to bypass cross-resistance among established 5-nitromidazole antiparasitic drugs.
doi:10.1128/AAC.50.1.344-347.2006
PMCID: PMC1346785  PMID: 16377707
6.  Rearranged Subtelomeric rRNA Genes in Giardia duodenalis 
Eukaryotic Cell  2005;4(2):484-486.
Giardia duodenalis has linear chromosomes capped with typical eukaryotic repeats [(TAGGG)n], subtelomeric rRNA genes, and telomere gene units. The absence of two closely associated NotI sites in the large-subunit rRNA gene was used as an indicator in hybridizations of one- and two-dimensional NotI-cleaved Giardia chromosome separations that some chromosomes carry only rearranged and, by deduction, nonfunctional rRNA genes.
doi:10.1128/EC.4.2.484-486.2005
PMCID: PMC549325  PMID: 15701810
7.  Orally Administered Giardia duodenalis Extracts Enhance an Antigen-Specific Antibody Response 
Infection and Immunity  2001;69(10):6503-6510.
We have identified novel adjuvant activity in specific cytosol fractions from trophozoites of Giardia isolate BRIS/95/HEPU/2041 (J. A. Upcroft, P. A. McDonnell, and P. Upcroft, Parasitol. Today, 14:281–284, 1998). Adjuvant activity was demonstrated in the systemic and mucosal compartments when Giardia extract was coadministered orally with antigen to mice. Enhanced antigen-specific serum antibody responses were demonstrated by enzyme-linked immunosorbent assay to be comparable to those generated by the “gold standard,” mucosal adjuvant cholera toxin. A source of adjuvant activity was localized to the cytosolic component of the parasite. Fractionation of the cytosol produced fraction pools, some of which, when coadministered with antigen, stimulated an enhanced antigen-specific serum response. The toxic component of conventional mucosal adjuvants is associated with adjuvant activity; therefore, in a similar way, the toxin-like attributes of BRIS/95/HEPU/2041 may be responsible for its adjuvanticity. Complete characterization of the adjuvant is under way.
doi:10.1128/IAI.69.10.6503-6510.2001
PMCID: PMC98786  PMID: 11553595
8.  Drug Susceptibility Testing of Anaerobic Protozoa 
A simple technique for routine, reproducible global surveillance of the drug susceptibility status of the anaerobic protozoa Trichomonas, Entamoeba, and Giardia is described. Data collected using this technique can be readily compared among different laboratories and with previously reported data. The technique employs a commercially available sachet and bag system to generate a low-oxygen environment and log2 drug dilutions in microtiter plates, which can be monitored without aerobic exposure, to assay drug-resistant laboratory lines and clinically resistant isolates. MICs (after 2 days) of 3.2 and 25 μM indicated metronidazole-sensitive and highly clinically resistant isolates of T. vaginalis in anaerobic assays, respectively. The aerobic MICs were 25 and >200 μM. MICs (1 day) of 12.5 to 25 μM were found for axenic lines of E. histolytica, and MICs for G. duodenalis (3 days) ranged from 6.3 μM for metronidazole-sensitive isolates to 50 μM for laboratory metronidazole-resistant lines. This technique should encourage more extensive monitoring of drug resistance in these organisms.
doi:10.1128/AAC.45.6.1810-1814.2001
PMCID: PMC90550  PMID: 11353630
9.  Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa 
Clinical Microbiology Reviews  2001;14(1):150-164.
The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated using laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.
doi:10.1128/CMR.14.1.150-164.2001
PMCID: PMC88967  PMID: 11148007
10.  Efficacy of New 5-Nitroimidazoles against Metronidazole-Susceptible and -Resistant Giardia, Trichomonas, and Entamoeba spp. 
The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via SRN1 and subsequent reactions, were assayed against the protozoan parasites Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica. Two metronidazole-sensitive lines and two metronidazole-resistant lines of Giardia and one line each of metronidazole-sensitive and -resistant Trichomonas were tested. All except one of the compounds were as effective or more effective than metronidazole against Giardia and Trichomonas, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against Entamoeba. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of Giardia and Trichomonas. However, some drugs were lethal to metronidazole-resistant Giardia and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.
PMCID: PMC89023  PMID: 9869568

Results 1-10 (10)