Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates

Abstract

Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates.

Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by ~100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites.

Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

Fig. 1

Analysis of VSP genes. VSP diversity in Giardia intestinalis assemblage A isolates. Black: WB (AI), Blue: AS98 (AII-1) and Red: AS175 (AII-2). An alignment-free method was used to compare the VSP repertoires. The VSPs from the three different assemblage A isolates form two major cluster with the AI VSPs separated from the AII VSPs

Fig. 2

Putative allelic sequence heterozygosity. Chromosome-wide distribution of putative allelic sequence heterozygosity (ASH). Red lines represent ASH in isolate AS175 (assemblage AII-1) and blue lines represent ASH in isolate AS98 (assemblage AII-2). Grey lines represents the inverted coding density, ie. more intense peaks represent less coding content and lower peaks represent higher coding content. Dots indicate the approximate chromosomal location of VSP genes (black) and HCMP genes (grey). Regions with higher content of ASH coincide with regions of lower coding content and often a VSP or HCMP gene. Regions with high coding content tend to be more devoid of ASH

Fig. 3

Close-up of ASH in a specific intergenic region on chromosome 2. The blue line represents ASH in isolate AS175 (assemblage AII-1) and grey line represents ASH in isolate AS98 (assemblage AII-2)

Fig. 4

Chromosome-wide distribution of single nucleotide polymorphisms. Chromosome-wide distribution of single nucleotide polymorphisms in 15 kb overlaping windows for each of the two isolates compared to WB. The blue lines represents the AS98 isolate compared to WB and the black lines represent AS175 compared to WB. The number of SNPs is shown on the y-axis and the chromosomal position on the x-axis

Fig. 5

Phylogenetic analysis of Bacteriocidal and Permeability Inducing-like (BPIL) proteins in Giardia. Amino acid maximum likelihood phylogeny based on 485 unambiguously aligned amino acid positions. The sequences are color coded according to isolates: G. intestinalis WB (red), G. intestinalis DH (orange), G. intestinalis GS (blue), G. intestinalis P15 (green), and G. muris (brown). Bootstrap support values >50 are shown. The tree is rooted on the branch leading to G. muris

Fig. 6

Localization of BPI-like proteins in Giardia. The BPI-like protein GL50803_16293 was epitope-tagged using a triple HA-tag and the fusion protein was localized to the ER in Giardia trophozoites using an anti-HA antibody