Genetic diversity in the metronidazole metabolism genes nitroreductases and pyruvate ferredoxin oxidoreductases in susceptible and refractory clinical samples of Giardia lamblia

Abstract

The effectiveness of metronidazole against the tetraploid intestinal parasite Giardia lamblia is dependent on its activation/inactivation within the cytoplasm. There are several activating enzymes, including pyruvate ferredoxin reductase (PFOR) and nitroreductase (NR) 1 which metabolize metronidazole into toxic forms, while NR2 on the other hand inactivates it. Metronidazole treatment failures have been increasing rapidly over the last decade, indicating genetic resistance mechanisms. Analyzing genetic variation in the PFOR and NR genes in susceptible and refractory Giardia isolates may help identify potential markers of resistance. Full length PFOR1, PFOR2, NR1 and NR2 genes from clinical culturable isolates and non-cultured clinical Giardia assemblage B samples were cloned, sequenced and single nucleotide variants (SNVs) were analyzed to assess genetic diversity and alleles. A similar ratio of amino acid changing SNVs per gene length was found for the NRs; 4.2% for NR1 and 6.4% for NR2, while the PFOR1 and PFOR2 genes had less variability with a ratio of 1.1% and 1.6%, respectively. One of the samples from a refractory case had a nonsense mutation which caused a truncated NR1 gene in one out of six alleles. Further, we found three NR2 alleles with frameshift mutations, possibly causing a truncated protein in two susceptible isolates. One of these isolates was homozygous for the affected NR2 allele. Three nsSNVs with potential for affecting protein function were found in the ferredoxin domain of the PFOR2 gene. The considerable variation and discovery of mutations possibly causing dysfunctional NR proteins in clinical Giardia assemblage B isolates, reveal a potential for genetic link to metronidazole susceptibility and resistance.

Keywords: Allele; Genetic diversity; Metronidazole; Nitroreductase; Nonsense mutation; Pyruvate ferredoxin oxidoreductase; Resistance; SNP; SNV.

Graphical abstract

Fig. 1

Cartoon presentations of NR1 and NR2 homology models. The ferredoxin (fd) domain is above, and iron-sulfur cluster is shown as a red-yellow cage within the fd domain. The larger NR domain is below. nsSNVs identified in the present study are depicted with orange color. Red color indicates nonsense mutations at positions 23, 37 and 151 from susceptible NR2 isolates, and a nonsense mutation leading to a premature stop codon at position 195 from a refractory NR1 isolate (positions highlighted by *). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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