Genomic diversity of the human intestinal parasite Entamoeba histolytica

Abstract

Background: Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome.

Results: The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually.

Conclusions: E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven.

Figure 1

Relationships among sequenced E. histolytica strains. Relationships were estimated using 3,696 polymorphic sites and two phylogenetic approaches, distance-based analysis (neighbor-joining) and maximum likelihood (ML), both as implemented in MEGA 5 [52]. ML used the General Time Reversible model of evolution, selected as best using the model testing function in MEGA 5, while neighbor-joining used the maximum composite likelihood model. Statistical support for distance and ML trees was evaluated using bootstrapping (1,000 replicates). The ML tree is shown. The tree is rooted by comparison with orthologous positions in E. dispar (data not shown) along the branch to the PVBM08B and PVBM08F strains. Bootstrap values from both types of phylogenetic analysis are shown in the following order: Distance/ML. An asterisk indicates where the bootstrap value is under 50%.

Figure 2

Evidence of historical recombination among E. histolytica. The proportion of 100,000 randomly sampled pairs of polymorphic sites showing 4 haplotypes calculated for 100 distance intervals, plotted against the median distance for each interval. The positive correlation is highly significant, Spearman's rho = 0.83 (P < 2.2 × 10^-16).

Figure 3

The pattern of polymorphism across scaffold DS571171. Polymorphic sites on scaffold DS571171 are represented by shaded squares: black for bases that match the HM-1:IMSS reference; light grey for bases that differ from the reference; dark grey for heterozygous positions; white for positions where no base was assigned. Three positions (marked with asterisks) had an additional variant base in one strain. Each row represents a strain in the following order from top to bottom: reference sequence (HM-1:IMSS), HM-1A, HM-1B, Rahman, 2592100, PVBM08B, PVBM08F, IULA:1092:1, HK-9, MS84-1373, MS27-5030. Sites in coding sequences are marked with black lines above the figure.

Figure 4

Copy number variation among E. histolytica genes. Coverage depth of a set of 6,228 E. histolytica genes relative to HM-1:IMSS in eight sequenced strains. The y-axis displays the ratio of a strain's RPKM to the average RPKM of HM-1A and HM-1B. The x-axis represents 6,228 annotated E. histolytica genes ordered by their amoebaDB gene ID. Ratios greater than 10 were truncated for display purposes. Grey bars represent results for all uniquely aligned reads, black bars for uniquely aligned reads with unique start positions on the reference genome (see Materials and methods).

Figure 5

Segmental duplication in the E. histolytica Rahman strain. High coverage across a region of scaffold DS571330 in the Rahman genome is confirmed by independently generated sequencing libraries (SOLiD and 454). The region encompasses seven genes and is flanked by repetitive elements. The black line represents coverage from Rahman SOLiD sequencing library, the dashed line represents coverage from Rahman 454 sequencing library, the grey line represents coverage from strain HM-1B SOLiD sequencing library, and is shown as a control. Coverage depth is displayed on a log scale in order to show both SOLiD and 454 data (where many fewer reads are generated) on the same plot.