Physiological aspects of nitro drug resistance in Giardia lamblia

Joachim Müller,Andrew Hemphill,Norbert Müller

doi:10.1016/j.ijpddr.2018.04.008

Joachim Müller, Andrew Hemphill + Show 1 more

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https://doi.org/10.1016/j.ijpddr.2018.04.008

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Abstract

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.

Highlights

According to a commonly accepted model, nitro compounds are activated by reduction yielding toxic intermediates, the electrons being provided by pyruvate oxidoreductase (POR)
In order to illustrate the resistance of the G. lamblia strain C4 derived from the wild-type WBC6, we determined the minimal inhibitory concentrations of three nitro compounds, namely MET, NTZ and
In order to see whether the results obtained on nitro reduction can be extended to metabolic processes involving other electron acceptors, we investigated oxygen consumption and resazurin reduction, both methods using intact cells

Summary

Introduction

Most of the studies compare resistant “model” strains generated in vitro with isogenic wildtype strains (Upcroft, 1998) These studies have revealed genome rearrangements (Upcroft et al, 1990, 1992) and profound transcriptional changes evidenced by differential analyses using microarrays followed by quantitative RT-PCR on selected transcripts (Müller et al, 2008) and strand-specific RNA sequencing (Ansell et al, 2017). In both studies, expression profiles of genes coding for variant surface proteins and for genes involved in oxido-reductions – amongst others - are altered the latter allegedly confirming this hypothesis

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