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

PLoS Pathog. 2022 Sep 27;18(9):e1010840. doi: 10.1371/journal.ppat.1010840. eCollection 2022 Sep.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Albendazole / chemistry
  • Albendazole / pharmacokinetics
  • Animals
  • Anthelmintics* / pharmacology
  • Biotransformation
  • Chromatography, Liquid
  • Cytochromes / metabolism
  • Flavins / metabolism
  • Giardia lamblia* / genetics
  • Giardia lamblia* / metabolism
  • Heme / metabolism
  • Humans
  • Iron
  • Metronidazole / pharmacology
  • Mixed Function Oxygenases / metabolism
  • Molecular Docking Simulation
  • RNA, Messenger / metabolism
  • Sulfones
  • Sulfoxides / metabolism
  • Superoxides
  • Tandem Mass Spectrometry
  • Trophozoites / metabolism
  • Xanthine Oxidase / metabolism
  • Xanthines

Substances

  • Anthelmintics
  • Cytochromes
  • Flavins
  • RNA, Messenger
  • Sulfones
  • Sulfoxides
  • Xanthines
  • Superoxides
  • Metronidazole
  • Heme
  • Iron
  • Mixed Function Oxygenases
  • Xanthine Oxidase
  • Albendazole

Grants and funding

This research was funded by Fondo SEP-Cinvestav, Grant Number FIDSC 2018/95 and by Fondo Sectorial Secretaría de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP–CONACYT) México Grant number A1-S-39422 to GOP and partially by Grant 282663 to ES. Funding was also provided by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (Grant Number 2018-04165) to SPR.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.