Genetic variation in metronidazole metabolism and oxidative stress pathways in clinical Giardia lamblia assemblage A and B isolates

Abstract

Purpose: Treatment-refractory Giardia cases have increased rapidly within the last decade. No markers of resistance nor a standardized susceptibility test have been established yet, but several enzymes and their pathways have been associated with metronidazole (MTZ) resistant Giardia. Very limited data are available regarding genetic variation in these pathways. We aimed to investigate genetic variation in metabolic pathway genes proposed to be involved in MTZ resistance in recently acquired, cultured clinical isolates. Methods: Whole genome sequencing of 12 assemblage A2 and 8 assemblage B isolates was done, to decipher genomic variation in Giardia. Twenty-nine genes were identified in a literature search and investigated for their single nucleotide variants (SNVs) in the coding/non-coding regions of the genes, either as amino acid changing (non-synonymous SNVs) or non-changing SNVs (synonymous). Results: In Giardia assemblage B, several genes involved in MTZ activation or oxidative stress management were found to have higher numbers of non-synonymous SNVs (thioredoxin peroxidase, nitroreductase 1, ferredoxin 2, NADH oxidase, nitroreductase 2, alcohol dehydrogenase, ferredoxin 4 and ferredoxin 1) than the average variation. For Giardia assemblage A2, the highest genetic variability was found in the ferredoxin 2, ferredoxin 6 and in nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase putative genes. SNVs found in the ferredoxins and nitroreductases were analyzed further by alignment and homology modeling. SNVs close to the iron-sulfur cluster binding sites in nitroreductase-1 and 2 and ferredoxin 2 and 4 could potentially affect protein function. Flavohemoprotein seems to be a variable-copy gene, due to higher, but variable coverage compared to other genes investigated. Conclusion: In clinical Giardia isolates, genetic variability is common in important genes in the MTZ metabolizing pathway and in the management of oxidative and nitrosative stress and includes high numbers of non-synonymous SNVs. Some of the identified amino acid changes could potentially affect the respective proteins important in the MTZ metabolism.

Keywords: drug metabolism; ferredoxin; genetic analysis; genetic diversity; metronidazole genes; resistance.

Figure 1

Heat map of proteins in the metabolism of metronidazole and oxidative/nitrosative stress management in Giardia assemblage B. Metronidazole (MTZ) passively diffuses through the trophozoite membrane and needs to be activated into toxic intermediates to execute its function as an antibiotic. Several theoretical intermediates exist for MTZ. MTZ can be activated either through the enzymes pyruvate: ferredoxin oxidoreductase (PFOR)-1 and/or 2 with ferredoxin (fd) as a co-factor, by nitroreductase-1 with flavin mononucleotide (FMN) as a co-factor or by thioredoxin reductase with the redox cofactor flavin adenine dinucleotide (FAD). MTZ may also be converted into an inert metabolite through the enzyme nitroreductase-2. The MTZ-NO formed during activation may react with O₂⁻ and create the reactive free radical molecule peroxynitrite (ONOO⁻). In order to remove toxic free radicals many enzymes exhibit protective functions, ie, thioredoxin peroxidase enzymes may convert the peroxynitrite to the harmless molecule nitrate (NO₃⁻). The free radical form of NO^· may be converted to nitrate by flavohemoprotein (O₂ dependent reaction). In the microaerophilic environment, Giardia is repeatedly exposed to the harmful O₂/O₂⁻. The O₂ may be metabolized to H₂O₂ through desulfoferredoxin (SOR) and further H₂O₂ is converted to H₂O by the thioredoxin peroxidase enzymes. O₂ may also be converted to H₂O by NADH oxidase enzymes or the O₂ scavenging enzyme flavoprotein. Other enzymes may cause free radicals in Giardia, ie, NADPH oxidoreductase putative is areactive oxygen species (ROS) contributor due to conversion of O₂ to a more reactive free radical form (O₂⁻). Another contributor to ROS is the reduced version of thioredoxin and it may initiate protein misfolding. The color gradation of the proteins represents the number of nsSNVs positions per length of each gene. *Thioredoxin reductase is active in both MTZ metabolism and reduction of the protein thioredoxin. **Flavohemoprotein is a suspected multicopy gene and the number of SNVs has not been defined and is not represented by color in the heatmap.

Figure 2

Alignment of the ferredoxins (Fd1-Fd6) and nitroreductase 1 and 2. The genetic variability is represented by alternative amino acids under the original protein sequences. Ferredoxin 5 was found to be an uncharacterized protein and is not annotated in the DHA2 and GSB genomes and is thus presented by the hypothetical sequence from Giardia WB. Only the ferredoxin domains in the N-terminus of the proteins are represented, and the nitro-flavin mononucleotide (FMN) reductase domain in the C-terminus of the NRs are not shown. The cysteine residues responsible for forming the iron-sulfur clusters are shaded in either black or magenta to represent how the residues connect. *end of the protein, +the protein has been cut and only the ferredoxin domain is shown.

Figure 3

Cartoon tube representation of homology models of Nitroreductase (NR) ferredoxin domains and ferredoxins (Fd) 1–6. Cysteine residues responsible for the binding of [Fe4-S4] iron–sulfur cluster are represented with sticks colored with dark gray and red for clusters 1 and 2, respectively. For the mutations caused by SNVs, the original amino acid residue is shown in stick and possible mutations are marked with one letter code. (A) NR1_A2 (grey), NR2_A2 (cyan), NR1_B (yellow) and NR2_B (brown) (B) Fd1_A2 (grey), Fd2_A2 (cyan), Fd1_B (yellow) and Fd2_B (brown) (C) Fd3_A2 (grey), Fd3_B (cyan). The extended C-terminus has been truncated from the figure and is marked with an asterisk. (D) Fd4_A2 (grey), Fd4_B (cyan). (E) Fd5_WB (grey) (F) Fd6_A2 (grey), Fd6_B (cyan).