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.

Perspectives on the Drug Discovery of Intestinal Protozoan Parasites

“…Furthermore, a sharp rise in refractory patients has also been documented in India [47]. In vitro studies have illustrated that the induction of resistance to benzimidazole is correlated with β-tubulin mutation, degradation of enzymes involed in glycolysis and arginine metabolism, and reduced mRNA expression of flavohemoglobin [36]. The detailed molecular mechanisms of resistance that lead to 5-nitroimidazole refractory Giardia infection as well as other types of resistance is not completely understood despite substantial efforts with laboratory as well as clinical strains.…”

“…Also, this drug is not recommended during pregnancy due to teratogenic effects in animals and may be in humans as well [17]. Trophozoites metabolize albendazole and result in the formation of toxic intermediates: albendazole sulfone and albendazole sulfoxide with the help of flavohemoglobin, a NADH oxidase (gNADHox) present inside the parasite (Figure 1) [36]. It has been established that the albendazole binds to the beta-tubulin of the parasite, and the presence of two amino acids (Glu-198 and Phe-200) in the beta-tubulin sequence renders this protozoan susceptible to benzimidazoles (Table 1).…”

Leading Paediatric Infectious Diseases—Current Trends, Gaps, and Future Prospects in Oral Pharmacotherapeutic Interventions

“…However, as with most of the infectious diseases, the cornerstone therapy for H. pylori in children has long been the traditional triple therapy consisting of the proton pump inhibitors, as well as antibiotics, namely amoxicillin, clarithromycin, and metronidazole that are also known to be the most common amongst drugs employed in the treatment of other bacterial infectious diseases. As such, these anti-infective agents were recorded as one of the most susceptible to microbial resistance [ 197 , 198 , 199 , 200 ]. In addition, the World Health Organization published a new recommendation for treating tuberculosis using a first-line regimen to shorten therapy from six to four months in efforts to reduce prolonged exposure whilst maintaining clinical efficacy [ 201 , 202 ].…”

Ultrastructural Alterations of the Human Pathogen Giardia intestinalis after Drug Treatment

“…Albendazole, a benzimidazole carbamate, exhibits activity against G. intestinalis , and trophozoites can metabolize this drug into its toxic intermediates: albendazole sulfone and albendazole sulfoxide. The conversion process involves a recently described flavohemoglobin present in the parasite [ 43 ]. Despite the fact that several microtubular structures constitute the parasite’s cytoskeleton, electron microscopy studies have shown that the drug specifically affects the ventral disc ( Figure 6 ) [ 44 ].…”

“…Furthermore, albendazole can potentially interfere with mitotic spindle development during cell division, particularly affecting the G2 phase of the G. intestinalis cell cycle [33]. In vitro studies have demonstrated the ability to induce albendazole resistance, which correlates with beta-tubulin mutation, modulation of enzymes involved in glycolysis and arginine metabolism, and decreased Giardia flavohemoglobin mRNA expression [43,45,46]. peripheral vesicles (P).…”