Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole

Abstract

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite. Author summary A major concern in the treatment of infectious diseases is the increased prevalence and distribution of drug resistance in microorganisms. In relation to giardiasis, there is an increase resistance to widespread, routinely used drugs, including albendazole (ABZ). Although G. duodenalis may convert ABZ to its mono-oxygenated form, albendazole sulfoxide (ABZSO), and further oxidize it to the less active albendazole sulfone (ABZSOO), the canonical molecular machineries for such transformations, namely cytochrome P450s (CYP450s) and flavin monooxygenases (FMOs), are not encoded in the genome of this parasite. In this work, we show that G. duodenalis flavohemoglobin (gFlHb) contributes to ABZ → ABZSO biotransformation at least via oxygenation, mediated by superoxide derived from its NADH oxidase activity, and that the mRNA expression of the respective gene is down-regulated in ABZ-resistant parasites, suggesting that this enzyme is inversely related to the ABZ-resistant phenotype. Consequently, gFlHb seems to play a novel function/mechanism in ABZ biotransformation, reinforcing its status as a novel therapeutic target against G. duodenalis, particulrly since it is absent from the human host.