Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.

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

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.

No difference in serum levels of B-cell activating receptor and antibodies against cytolethal distending toxin B and flagellin in post-infectious irritable bowel syndrome and chronic fatigue syndrome after Giardia infection.

Background and Aim: Functional gastrointestinal disorders (FGIDs) and chronic fatigue syndrome (CFS) frequently occur as comorbid conditions to each other. A shared etiology of these syndromes has been proposed because of their shared symptomatology and triggering by infections. Antibodies against the bacterial antigens cytolethal distending toxin B (CdtB) and flagellin have been proposed to be biomarkers of irritable bowel syndrome (IBS), especially diarrhea-predominant IBS (IBS-D). It is unknown if they may also be associated with comorbid conditions such as CFS. On the other hand, elevated level of B-cell activating factor (BAFF) has been associated with CFS and inflammatory bowel disease (IBD) and subjective food intolerance. Methods: We evaluated serum levels of anti-flagellin and anti-CdtB using an in-house enzyme-linked immunosorbent assay (ELISA) and BAFF with a commercially available ELISA kit in a cohort of patients who developed fatigue syndromes and/or FGIDs after Giardia infection, by comparing them with healthy controls without these conditions. Results: We did not find significant differences in circulating BAFF, anti-CdtB, or anti-flagellin antibody levels in these patient groups compared to healthy controls. Therefore, our results do not support a role for BAFF, anti-CdtB, or anti-flagellin antibodies as universal biomarkers for IBS or CFS. Conclusion: BAFF, anti-CdtB, or anti-flagellin antibodies cannot be considered as universal biomarkers for IBS or CFS.

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.

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

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.

Genetic variation in potential Giardia vaccine candidates cyst wall protein 2 and α1-giardin.

Giardia is a prevalent intestinal parasitic infection. The trophozoite structural protein a1-giardin (a1-g) and the cyst protein cyst wall protein 2 (CWP2) have shown promise as Giardia vaccine antigen candidates in murine models. The present study assesses the genetic diversity of a1-g and CWP2 between and within assemblages A and B in human clinical isolates. a1-g and CWP2 sequences were acquired from 15 Norwegian isolates by PCR amplification and 20 sequences from German cultured isolates by whole genome sequencing. Sequences were aligned to reference genomes from assemblage A2 and B to identify genetic variance. Genetic diversity was found between assemblage A and B reference sequences for both a1-g (90.8% nucleotide identity) and CWP2 (82.5% nucleotide identity). However, for a1-g, this translated into only 3 amino acid (aa) substitutions, while for CWP2 there were 41 aa substitutions, and also one aa deletion. Genetic diversity within assemblage B was larger; nucleotide identity 92.0% for a1-g and 94.3% for CWP2, than within assemblage A (nucleotide identity 99.0% for a1-g and 99.7% for CWP2). For CWP2, the diversity on both nucleotide and protein level was higher in the C-terminal end. Predicted antigenic epitopes were not affected for a1-g, but partially for CWP2. Despite genetic diversity in a1-g, we found aa sequence, characteristics, and antigenicity to be well preserved. CWP2 showed more aa variance and potential antigenic differences. Several CWP2 antigens might be necessary in a future Giardia vaccine to provide cross protection against both Giardia assemblages infecting humans.

Human Memory CD4+ T Cell Immune Responses against Giardia lamblia.

The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, TH17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4(+) T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4(+) effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4(+) EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4(+) T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4(+) EM T cell response of which IL-17A production seems to be an important component.