<p>Genetic variation in metronidazole metabolism and oxidative stress pathways in clinical<em> Giardia lamblia</em> assemblage A and B isolates</p>

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.

<p>Genetic Diversity of the Flavohemoprotein Gene of Giardia lamblia: Evidence for High Allelic Heterozygosity and Copy Number Variation</p>

“… 44 Specific primers for the two orthologs of gFlHb of sub-assemblage AII and assemblage B were designed in Geneious Prime ® 2019.0.3 (Biomatters Ltd., Auckland, New Zealand) (see Table 1 ) based on conserved pre-and post-CDS regions of the gFlHb gene represented in twelve assemblage A and eight assemblage B whole genome sequenced isolates previously presented in a former study. 42 Each 25 µL PCR reaction contained 1X Q5 Reaction Buffer (cat. nr: B90276, New England BioLabs (NEB) Ipswich, MA, USA), 2 mM MgCl 2 (included in buffer), 0.2 mM dNTP’s (catalog nr N0447L, NEB), 0.5 U Q5 High-Fidelity DNA Polymerase (catalog nr M0491L NEB) 0.2 µM each of forward and reverse primer, 1 µL template DNA and nuclease-free water up to 25 µL.…”

“…43 Collection, DNA extraction, concentration measurements and Illumina-based whole genome sequencing were carried out as previously stated. 42 No clinical data have been collected from the clinical samples of Giardia. assemblage AII (DHA2_154000) and B (GSB_151570) were amplified from genomic DNA by PCR.…”

“…44 Specific primers for the two orthologs of gFlHb of sub-assemblage AII and assemblage B were designed in Geneious Prime ® 2019.0.3 (Biomatters Ltd., Auckland, New Zealand) (see Table 1) based on conserved pre-and post-CDS regions of the gFlHb gene represented in twelve assemblage A and eight assemblage B whole genome sequenced isolates previously presented in a former study. 42 Amplified PCR products were run on 1% Agarose gels stained with GelGreen ® Nucleic Acid Stain (catalog nr: 41,005, Biotium, San Fransisco, CA, USA) and positive bands were cut from the gel using blue light illuminator (Serva, Heidelberg, Germany) and extracted using Wizard SV gel and PCR Clean-Up System (catalog nr: A9282, Promega, Madison, WI, USA) according to manufacturer's descriptions, the only exception was using 70°C nucleasefree water in the last elution step. Concentrations of the PCR products were measured using Quantus™ Fluorometer (catalog Nr: E6150, Promega).…”

“…[35][36][37][38][39][40][41] In a recent study on the genetic diversity of genes involved in MTZ induced oxidative and nitrosative stress management, we found indications that gFlHb genes may not only present allelic variation, but also be present at variable copy numbers per haploid genome. 42 The current study is a follow-up study of this recent published article. 42 As gFlHb may play a role in the ability of Giardia to tolerate MTZ, we performed this study to further explore the copy number variability and the allelic diversity of gFlHb in G. lamblia strains representing recent sub-assemblage AII and assemblage B isolates.…”

“… 42 The current study is a follow-up study of this recent published article. 42 As gFlHb may play a role in the ability of Giardia to tolerate MTZ, we performed this study to further explore the copy number variability and the allelic diversity of gFlHb in G. lamblia strains representing recent sub-assemblage AII and assemblage B isolates.…”