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>Genetic Diversity of the Flavohemoprotein Gene of Giardia lamblia: Evidence for High Allelic Heterozygosity and Copy Number Variation
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Genetic Diversity of the Flavohemoprotein Gene of Giardia lamblia: Evidence for High Allelic Heterozygosity and Copy Number Variation
Purpose:The flavohemoprotein (gFlHb) in Giardia plays an important role in managing nitrosative and oxidative stress, and potentially also in virulence and nitroimidazole drug tolerance. The aim of this study was to analyze the genetic diversity of gFlHb in Giardia assemblages A and B clinical isolates.Methods:gFlHb genes from 20 cultured clinical Giardia isolates were subjected to PCR amplification and cloning, followed by Sanger sequencing. Sequences of all cloned PCR fragments from each isolate were analyzed for single nucleotide variants (SNVs) and compared to genomic Illumina sequence data. Identical clone sequences were sorted into alleles, and diversity was further analyzed. The number of gFlHb gene copies was assessed by mining PacBio de novo assembled genomes in eight isolates. Homology models for assessment of SNV's potential impact on protein function were created using Phyre2.Results:A variable copy number of the gFlHb gene, between two and six copies, depending on isolate, was found. A total of 37 distinct sequences, representing different alleles of the gFlHb gene, were identified in AII isolates, and 41 were identified in B isolates. In some isolates, up to 12 different alleles were found. The total allelic diversity was high for both assemblages (0.9) and was coupled with a nucleotide diversity of 0.01. The genetic variation (SNVs per CDS length) was 4.8% in sub-assemblage AII and 5.4% in assemblage B. The number of non-synonymous (ns) SNVs was high in gFIHb of both assemblages, 1.6% in A and 3.0% in B, respectively. Some of the identified nsSNV are predicted to alter protein structure and possibly function.Conclusion:In this study, we present evidence that gFlHb, a putative protective enzyme against oxidative and nitrosative stress in Giardia, is a variable copy number gene with high allelic diversity. The genetic variability of gFlHb may contribute metabolic adaptability against metronidazole toxicity.? 2020 Saghaug et al.
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机译：目的：Giardia的黄萎病蛋白（GFLHB）在管理亚硝化和氧化应激方面发挥着重要作用，并且可能也具有毒力和硝基咪唑耐药性。本研究的目的是分析GlhHB在Giardia组合A和B临床分离株中的遗传多样性。方法：从20种培养的临床Giardia分离物中的GflHB基因进行PCR扩增和克隆，然后进行Sanger测序。分析来自每种分离物的所有克隆的PCR片段的序列，用于单核苷酸变体（SNV）并与基因组Illumina序列数据进行比较。将相同的克隆序列分类为等位基因，进一步分析多样性。通过在8个分离株中采用PACBIO DE Novo组装的基因组来评估GFLHB基因拷贝的数量。使用PHYRE2创建了用于评估SNV潜在影响对蛋白质功能的同源模型。结果：发现，根据分离物，GFLHB基因的可变拷贝数，两到六份之间。在AII分离株中鉴定出代表GFLHB基因不同等位基因的37种不同的序列，并在B分离物中鉴定41个。在一些分离物中，找到了最多12个不同的等位基因。总体的多样性为组合物（> 0.9）高，并与核苷酸多样性相结合<0.01。亚组合AII的遗传变异（每CDS长度的SNV）为4.8％，组合B中的5.4％。在两个组件的GFIHB中，非同义（NS）SNV的数量高，1.6％和3.0％ b分别。预计一些已鉴定的NSSNV可以改变蛋白质结构和可能的功能。结论：在这项研究中，我们提出了一种证据表明，GFLHB是GFLHB，一种针对Giardia中的氧化和亚硝酸盐胁迫的证据是具有高等位基因的可变拷贝数基因。 GFLHB的遗传变异可能导致对甲硝唑毒性的代谢适应性。 2020 Saghaug等人。
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