Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites

Abstract

Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.

Fig. 1.

Physical characteristics of E. hellem, E. romaleae, E. intestinalis, and E. cuniculi genomes. Chromosomes I–XI are numbered according to their respective sizes in E. cuniculi (9) and are shown to scale. Chromosome IX is fragmented in assemblies of E. hellem and E. romaleae (as indicated by IXa and IXb in E. hellem and IXa–IXc in E. romaleae), but there is no evidence supporting an actual physical fragmentation of this chromosome in either species.

Fig. 2.

Genomic context of HGT-acquired genes in E. hellem and E. romaleae. The genes in question that are unique to E. hellem and E. romaleae are shown in red, and syntenic genes that are common to all four completed Encephalitozoon genomes are shown in cyan. The rRNA genes are shown in orange, unknown ORFs displaying no synteny are shown in beige, and subtelomeric genes unique to E. cuniculi are shown in green. Decayed folate-related pseudogenes in E. romaleae are indicated by a ψ-sign.

Fig. 3.

Hybrid origin of the folate metabolic pathways in E. hellem and E. romaleae. Genes vertically inherited in E. hellem, E. romaleae, E. intestinalis, and E. cuniculi are shown in blue. Genes acquired by HGT in the lineage leading to E. hellem and E. romaleae are shown in orange. Functions for which specific genes have not been attributed in E. hellem or E. romaleae are shown in dark gray. Arrows denote the directions of the enzymatic reactions. Asterisks indicate genes that underwent pseudogenization in E. romaleae. FT, folate transporter; GK, guanylate kinase; PK, pyruvate kinase; SHMT, serine hydroxymethyltransferase. Note that the putative FT function attributed to homologs of ECU11_1600 (unique to Encephalitozoon species) is uncertain, and that folate transport often is carried out by miscellaneous ATP transporters. The presence of all these genes in the E. hellem and E. romaleae genomes was confirmed using PCR and sequencing.

Fig. 4.

Bayesian phylogenetic trees of three proteins, PRT (A), GTPCH (B), and FPGS (C), involved in the folate metabolic pathways in E. hellem and E. romaleae (shown in white on black) but absent from other microsporidians. Numbers at nodes representing Bayesian posterior probabilities (Left) and bootstrap proportions (Right) are indicated when higher than 0.8 and 70%, respectively. The scale bar corresponds to the estimated number of amino acid substitutions per site. All trees are shown unrooted.