Contiguous and complete assemblies of Blastocystis gut microbiome-associated protists reveal evolutionary diversification to host ecology

Abstract

Blastocystis, an obligate host-associated protist, is the most common microbial eukaryote in the human gut, and is widely distributed across vertebrate hosts. The evolutionary transition of Blastocystis from its free-living stramenopile ancestors to a radiation of host-associated organisms is poorly understood. To explore this, we cultured and sequenced eight strains representing the significant phylogenetic diversity of the genus using long-read, short-read, and Hi-C DNA sequencing, alongside gene annotation and RNA sequencing. Comparative genomic analyses reveal significant variation in gene content and genome structure across Blastocystis. Notably, three strains from herbivorous tortoises, phylogenetically distant from human subtypes, have markedly larger genomes with longer introns and intergenic regions, and retain canonical stop codons absent in the human-associated strains. Despite these genetic differences, all eight isolates exhibit gene losses linked to the reduced cellular complexity of Blastocystis, including losses of cilia and flagella genes, microtubule motor genes, and signal transduction genes. Isolates from herbivorous tortoises contain higher numbers of plant carbohydrate-metabolizing enzymes, suggesting that, like gut bacteria, these protists ferment plant material in the host gut. We find evidence that some of these carbohydrate-metabolizing enzymes were horizontally acquired from bacteria, indicating that horizontal gene transfer is an ongoing process in Blastocystis that has contributed to host-related adaptation. Together, these results highlight substantial genetic and metabolic diversity within the Blastocystis genus, indicating that different lineages of Blastocystis have varied ecological roles in the host gut.

Figure 1.

Phylogenetic relationships and genome assembly characteristics of Blastocystis. (A) 18S rRNA maximum likelihood (ML) phylogeny of Blastocystis subtypes, with Proteromonas lacertae as an outgroup. Support values are 1000 replicates of ultrafast bootstrap and SH-aLRT tests (Guindon et al. 2010; Hoang et al. 2018). Strains in red were sequenced in this study. (B) 13-gene concatenation ML phylogeny for all strains and species with sequenced genomes used in this study. Important evolutionary transitions are highlighted. Images represent cellular morphology of Cafeteria burkhardae, Proteromonas lacertae, and Blastocystis. (C) Assembly statistics of Blastocystis strains sequenced in this work compared with previously sequenced and annotated genomes. Stop-codon aware and not stop codon aware designations refer to gene annotation methodologies that take into consideration that Blastocystis genes can lack stop codons or do not consider this, respectively. The ST7 N50 is a gapped scaffold N50 generated with Sanger sequencing scaffolding.

Figure 2.

Genomic features differ across Blastocystis strains. (A) Intron lengths and (B) intergenic lengths in tortoise isolates (left) and human isolates (right). (C) Sequence motif enriched adjacent to transcript ends in ST1, ST3, and ST4. Motif generated with STREME (Bailey 2021). This motif is referred to as “downstream gene element” (DGE). (D) Enrichment (log[observed/expected]) of transcripts with downstream conserved element within 30 bp upstream of or downstream from transcript end, normalized by expected DGE occurrence given GC content.

Figure 3.

Gene families lost or reduced in all Blastocystis strains. See Supplemental Table S2 for GO categories and InterPro domains used for defining categories.

Figure 4.

Clade-specific gene family expansions across Blastocystis, identified with InterProScan.

Figure 5.

Horizontal gene transfer across Blastocystis evolution. (A) Estimated time of transfer for 40 horizontally transferred genes in Blastocystis shared between more than one species or strain. These data include previously identified and validated cases originally identified in Eme et al. (2017) and newly identified transfer events. (B) Gene phylogeny of tortoise isolate alpha-rhamnosidase genes and 588 homologous proteins. Support values indicate 1000 replicates of SH-aLRT test and ultrafast bootstraps (UFboot). Branches with support values <95 for UFboot or <80 for SH-aLRT are collapsed.