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

Christina S Saghaug,1,2 Christian Klotz,3 Juha P Kallio,4 Toni Aebischer,3 Nina Langeland,1,2,5 Kurt Hanevik1 1Department of Clinical Science, University of Bergen, Bergen, Norway; 2Norwegian National Advisory Unit on Tropical Infectious Diseases, Department of Medicine, Haukeland University Hospital, Bergen, Norway; 3Department of Infectious Diseases, Unit 16 Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany; 4Department of Biomedicine, University of Bergen, Bergen, Norway; 5Department of Medicine, Haraldsplass Deaconess Hospital, Bergen, NorwayCorrespondence: Christina S SaghaugDepartment of Clinical Science, University of Bergen, 8th Floor, Lab-Building, Bergen N-5021, NorwayTel +47 90 13 24 14Email christina.saghaug@uib.noPurpose: 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 wer