Giardia lamblia is a protozoan parasite that causes giardiasis, a diarrhoeal disease affecting humans and various animal species. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for treatment of giardiasis. Nitroreductases such as GlNR1 and GlNR2 may play a role in activation or inactivation of these drugs. The aim of this work is to characterise these two enyzmes using functional assays. For respective analyses recombinant analogues from GlNR1 and GlNR2 were produced in Escherichia coli. E. coli expressing GlNR1 and GlNR2 alone or together were grown in the presence of nitro compounds. Furthermore, pull-down assays were performed using HA-tagged GlNR1 and GlNR2 as baits. As expected, E. coli expressing GlNR1 were more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions whereas E. coli expressing GlNR2 were susceptible to neither drug. Interestingly, expression of both nitroreductases gave the same results as expression of GlNR2 alone. In functional assays, both nitroreductases had their strongest activities on the quinone menadione (vitamin K3) and FAD, but reduction of nitro compounds including the nitro drugs metronidazole and nitazoxanide was clearly detected. Full reduction of 7-nitrocoumarin to 7-aminocoumarin was preferentially achieved with GlNR2. Pull-down assays revealed that GlNR1 and GlNR2 interacted in vivo forming a multienzyme complex. These findings suggest that both nitroreductases are multifunctional. Their main biological role may reside in the reduction of vitamin K analogues and FAD. Activation by GlNR1 or inactivation by GlNR2 of nitro drugs may be the consequence of a secondary enzymatic activity either yielding (GlNR1) or eliminating (GlNR2) toxic intermediates after reduction of these compounds.

For over 50 years, metronidazole and other nitro compounds such as nitazoxanide have been used as a therapy of choice against giardiasis and more and more frequently, resistance formation has been observed. Model systems allowing studies on biochemical aspects of resistance formation to nitro drugs are, however, scarce since resistant strains are often unstable in culture. In order to fill this gap, we have generated a stable metronidazole- and nitazoxanide-resistant Giardia lamblia WBC6 clone, the strain C4. Previous studies on strain C4 and the corresponding wild-type strain WBC6 revealed marked differences in the transcriptomes of both strains. Here, we present a physiological comparison between trophozoites of both strains with respect to their ultrastructure, whole cell activities such as oxygen consumption and resazurin reduction assays, key enzyme activities, and several metabolic key parameters such as NAD(P)+/NAD(P)H and ADP/ATP ratios and FAD contents. We show that nitro compound-resistant C4 trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and resazurin reduction rates, lower ornithine-carbamyl-transferase activity, reduced FAD and NADP(H) pool sizes and higher ADP/ATP ratios than wildtype trophozoites. The present results suggest that resistance formation against nitro compounds is correlated with metabolic adaptations resulting in a reduction of the activities of FAD-dependent oxidoreductases.

The anaerobic parasite Giardia lamblia, causative agent of persistent diarrhea, contains a family of nitroreductase genes most likely acquired by lateral transfer from anaerobic bacteria or archaebacteria. Two of these nitroreductases, containing a ferredoxin domain at their N-terminus, NR1, and NR2, have been characterized previously. Here, we present the characterization of a third member of this family, NR3. In functional assays, recombinant NR1 and NR3 reduced quinones like menadione and the antibiotic tetracycline, and-to much lesser extents-the nitro compound dinitrotoluene. Conversely, recombinant NR2 had no activity on tetracycline. Escherichia coli expressing NR3 were less susceptible to tetracycline, but more susceptible to the nitro compound metronidazole under semi-aerobic growth conditions. G. lamblia overexpressing NR1 and NR3, but not lines overexpressing NR2, are more susceptible to the nitro drug nitazoxanide. These findings suggest that NR3 is an active quinone reductase with a mode of action similar to NR1, but different from NR2. The biological function of this family of enzymes may reside in the use of xenobiotics as final electron acceptors. Thereby, these enzymes may provide at least two evolutionary advantages namely a higher potential to recycle NAD(P) as electron acceptors for the (fermentative) energy and intermediary metabolism, and the possibility to inactivate toxic xenobiotics produced by microorganisms living in concurrence inside the intestinal habitat.

As there is a continuous need for novel anti-infectives, the present study aimed to fuse two modes of action into a novel 3-nitroimidazo[1,2-b]pyridazine scaffold to improve antiparasitic efficacy. For this purpose, we combined known structural elements of phosphodiesterase inhibitors, a target recently proposed for Trypanosoma brucei and Giardia lamblia, with a nitroimidazole scaffold to generate nitrosative stress. The compounds were evaluated in vitro against a panel of protozoal parasites, namely Giardia lamblia, Trypanosoma brucei, T. cruzi, Leishmania infantum and Plasmodium falciparum and for cytotoxicity on MRC-5 cells. Interestingly, selective sub-nanomolar activity was obtained against G. lamblia, and by testing several analogues with and without the nitro group, it was shown that the presence of a nitro group, but not PDE inhibition, is responsible for the low IC50 values of these novel compounds. Adding the favourable drug-like properties (low molecular weight, cLogP (1.2-4.1) and low polar surface area), the key compounds from the 3-nitroimidazo[1,2-b]pyridazine series can be considered as valuable hits for further anti-giardiasis drug exploration and development.

The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea. Current treatments are based on nitro drugs, especially metronidazole. Nitro compounds are activated by reduction, yielding toxic intermediates. The enzymatic systems responsible for this activation are not completely understood. By fractionating cell free crude extracts by size exclusion chromatography followed by mass spectrometry, enzymes with nitroreductase (NR) activities are identified. The protein encoded by ORF 17150 found in two pools with NR activities is overexpressed and characterized. In pools of fractions with main NR activities, previously-known NRs are identified, as well as a previously uncharacterized protein encoded by ORF 17150. Recombinant protein 17150 is a flavoprotein with NADPH-dependent quinone reductase and NR activities. Besides a set of previously identified NRs, we have identified a novel enzyme with NR activity.

Giardia lamblia, a causative agent of persistent diarrhea in humans, domestic animals, and cattle, is usually treated with nitro compounds. Consequently, enzymes involved in anaerobic nitro reduction have been investigated in detail as potential targets. Their role within the normal metabolic context is, however, not understood. Using 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, we analyzed the metabolomes of G. lamblia trophozoites overexpressing three nitroreductases (NR1-NR3) and thioredoxin reductase (TrxR), most likely a scavenger of reactive oxygen species, as suggested by the results published in this study. We compared the patterns to convenient controls and to the situation in the nitro drug resistant strain C4 where NR1 is downregulated. We identified 27 metabolites in G. lamblia trophozoites. Excluding metabolites of high variability among different wildtype populations, only trophozoites overexpressing NR1 presented a distinct pattern of nine metabolites, in particular arginine catabolites, differing from the respective controls. This pattern matched a differential pattern between wildtype and strain C4. This suggests that NR1 interferes with arginine and thus energy metabolism. The exact metabolic function of NR1 (and the other nitroreductases) remains to be elucidated.

Giardia lamblia is a causative agent of persistent diarrhoea widespread in regions with low hygienic standards. Laboratory research is based on cloned lines issuing from various patient isolates typed in the late 1980s and 90s using restriction analysis and serology. In the present study, we compared the well-characterized strain WBC6 with another clone of the parent WB isolate termed WBA1 and with a clone from another isolate, GS/M-83-H7, using shotgun mass spectrometry proteomics. We identified 398 proteins differentially expressed between the GS and both WB isolates and 97 proteins differentially expressed between the two WB isolates. We investigated the expression levels of the predominant variant-specific surface proteins (VSPs) in each clone and matched the previously described major VSPs of each strain to the corresponding open reading frame sequences identified by whole-genome sequencing efforts. Furthermore, since the original WB isolate comes from a patient treated with metronidazole, we compared the susceptibilities of the strains to nitro compounds, as well the expression levels of enzymes involved in nitro reduction and on the corresponding enzyme activities and found distinct differences between the three strains.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the severe zoonotic disease alveolar echinococcosis. New treatment options are urgently needed. Disulfiram and dithiocarbamates were previously shown to exhibit activity against the trematode Schistosoma mansoni. As both parasites belong to the platyhelminths, here we investigated whether these compounds were also active against E. multilocularis metacestode vesicles in vitro. We used an in vitro drug-screening cascade for the identification of novel compounds against E. multilocularis metacestode vesicles with disulfiram and 51 dithiocarbamates. Five compounds showed activity against E. multilocularis metacestode vesicles after five days of drug incubation in a damage marker release assay. Structure-activity relationship analyses revealed that a S-2-hydroxy-5-nitro benzyl moiety was necessary for anti-echinococcal activity, as derivatives without this group had no effect on E. multilocularis metacestode vesicles. The five active compounds were further tested for potential cytotoxicity in mammalian cells. For two compounds with low toxicity (Schl-32.315 and Schl-33.652), IC50 values in metacestode vesicles and IC50 values in germinal layer cells were calculated. The compounds were not highly active on isolated GL cells with IC50 values of 27.0 ± 4.2 µM for Schl-32.315 and 24.7 ± 11.5 µM for Schl-33.652, respectively. Against metacestode vesicles, Schl-32.315 was not very active either with an IC50 value of 41.6 ± 3.2 µM, while Schl-33.652 showed a low IC50 of 4.3 ± 1 µM and should be further investigated in the future for its activity against alveolar echinococcosis.