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.

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power.

BACKGROUND: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. METHODS: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. RESULTS: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. CONCLUSION: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

Testing the impact of Whole Genome Amplification on genome comparison using the polyploid flagellated Giardia duodenalis as a model.

The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12 Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1 ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.

Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis.

The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.

Characterization of telomeres and telomerase from the single-celled eukaryote Giardia intestinalis.

The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes.

Porcine ubiquitin-like 5 (UBL5) gene: genomic organization, polymorphisms, mRNA cloning, splicing variants and association study.

Ubiquitin-like 5 (UBL5), which is supposed to be involved in regulation of feed intake, energy metabolism, obesity and type 2 diabetes, is located at position 62.1 cM on the pig chromosome 2 region harbouring quantitative trait loci for carcass and meat quality. The 4,354 bp genomic sequence (FR798948) of the porcine gene encompassing the promoter and entire gene was cloned by polymerase chain reaction. Comparative sequencing revealed 13 polymorphisms in noncoding regions. Synthesis of full-length cDNA sequences using rapid amplification of 5' and 3' ends showed three splice variants. Variants 1 and 2 differ in transcription length for the untranslated part of exon 1 with deduced protein of 73 amino acid (aa) residues and 100 % identities between human, mouse and other species. Variant 3, with 4 bp deletion at the 3' end of exon 2, encodes a truncated protein with 28 aa residues. In a Wild boar×Meishan F2 population (n = 334) with 47 recorded traits, loci FR798948:g.2788G>A and FR798948:g.2141T>C were associated at nominal P < 0.05 with fat deposition, growth and fattening and muscling but after adjustment for multiple testing (Benjamini and Hochberg, J R Stat Soc B 57:289-300, 1995) only eight fat deposition traits showed suggestive association with FR798948:g.2788G>A at adjusted P < 0.10. In a Meishan×Large White (MLW) cross (n = 562) with six trait records available, FR798948:g.2141T>C showed suggestive association with growth (adjusted P = 0.0690). As association mapping conducted in the outbred MLW population is more precise than in the three generation F2 population the UBL5 gene tends to be associated with growth rather than with fat accretion.