Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization

Abstract

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high-quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization. Given Legionella infection is known to alter its host's cell cycle, to exploit the host's organelles, and to modulate the host's metabolism in its favor, these changes in chromatin organization may partly be related to mechanisms of host control during Legionella infection.

Figure 1.

Assembly statistics for A. castellanii genomes. Comparison of genome assemblies for strains C3 and Neff-v2 versus the previous Neff-v1 genome assembly (Clarke et al. 2013). (A) Cumulative length plot showing the relationship between the number of contigs (largest to smallest) and length of the assembly. (B) General continuity metrics. (C) BUSCO statistics showing the status of universal single-copy orthologs in eukaryotes for each assembly. (D) Circos plot showing syntenic blocks for all scaffolds of A. castellanii strains C3 and Neff assemblies >50 kb.

Figure 2.

Numbers of strain-specific and shared orthologous groups in the genomes of A. castellanii strains C3 and Neff. Orthology inference was conducted with both Broccoli and OrthoFinder. Dictyostelium discoideum, Physarum polycephalum, and Vermamoeba vermiformis were used as outgroups to improve accuracy of orthogroup inference.

Figure 3.

Spatial organization of the A. castellanii genome. (A, top) Whole-genome Hi-C contact maps of the Neff (left) and C3 (right) genomes, with a magnification of the three largest scaffolds. The genomes are divided into 16-kb bins, and each pixel represents the contact intensity between a pair of bins. Each scaffold is visible as a red square on the diagonal. In both strains, there is an enrichment of interscaffold contacts toward telomeres, suggesting a spatial clustering of telomeres, as shown on the model in the right margin. (Bottom) 4C-like representation of spatial contacts between rDNA and the rest of the genome. Scaffolds are delimited by gray vertical lines. Contacts of all rDNAs are enriched toward telomeres. The genomic position of the 18S and 28S genes is highlighted with triangles on the top panel, and the occurrences of 8S rDNA sequences are shown with vertical red lines on the bottom panel. (B) High-resolution contact map for a region of the C3 genome showing chromatin loops detected by Chromosight as blue circles. (C) Size distribution of chromatin loops detected in the C3 strain.

Figure 4.

Changes in trans-chromosomal contacts between A. castellanii chromosomes following L. pneumophila infection. (A) Average contact change during infection between each pair of chromosomes. Chromosome lengths are shown below the interaction matrix, with the chromosome bearing 18S and 28S rDNA highlighted in green. (B) Representative intertelomeric contacts between a pair of chromosomes (C3 scaffolds 3 and 11). The average intertelomeric contact profile generated from all pairs of chromosomes is shown as a pileup. The log ratio between the infected (I) and uninfected (U) profiles is shown as a ratio (right).