Genetic variability of proteins involved in metronidazole metabolism and detoxification in Giardia lamblia

Abstract

Metronidazole (MTZ) treatment-refractory Giardia lamblia infections is an emerging global problem. Recent studies show that prevalence rates of refractory infections are 10-20 % and can be as high as 46 %. The recent global increase of treatment refractory Giardia infections, suggest an emergence of strains with certain genetic inheritable traits. Up until now, no specific genetic marker of MTZ-resistance has been discovered in Giardia and most in vitro studies have utilized laboratory-derived sub-assemblage AI isolates, which rarely infects humans. The overall aim of this thesis was to investigate and analyze single nucleotide variants (SNVs) in proteins that are responsible for activation or inactivation of MTZ, and involved in the detoxification of reactive oxidative species and reactive nitrosative species in clinical isolates of Giardia. In this thesis we address the presence of genetic variation, especially amino acid altering, non-synonymous(ns) SNVs, of 29 genes. The variation was identified by Illumina whole genome sequencing of 20 recent, clinical, cultured sub-assemblage AII and assemblage B isolates, while cloning and Sanger sequencing was performed to identify alleles of the three genes flavohemoprotein (gFlHb), nitroreductase (NR) 1 and 2, the two latter were explored in eight non-cultured clinical isolates as well. Further, gFlHb was investigated for its potential variable copy number by mining PacBio de novo assembled genomes in eight isolates. Our findings disclose high frequencies of genetic variations in many of the 29 genes, where nsSNVs in assemblage B were especially high in ferredoxins (fds), NR1 and NR2, thioredoxin peroxidase and NADH oxidase. gFlHb was found to be a variable copy number gene (2-6 copies/isolate) with high genetic variation. Moreover, we identified truncated NR1 alleles in one MTZ refractory isolate, while two susceptible isolates, one homozygotic, had truncated NR2 alleles. The considerable genetic variation, coupled with findings of variable copies of gFlHb and dysfunctional NR proteins in recent clinical isolates of Giardia show a potential for identification of genetic markers for MTZ susceptibility/resistance. Future studies will help to decipher the effects of the mutations and could include functional assays and obtaining samples from a larger, more well-defined patient cohort.