Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania

Abstract

Many high-quality genomes are available for dixenous (two hosts) trypanosomatid species of the genera Trypanosoma, Leishmania, and Phytomonas, but only fragmentary information is available for monoxenous (single-host) trypanosomatids. In trypanosomatids, monoxeny is ancestral to dixeny, thus it is anticipated that the genome sequences of the key monoxenous parasites will be instrumental for both understanding the origin of parasitism and the evolution of dixeny. Here, we present a high-quality genome for Leptomonas pyrrhocoris, which is closely related to the dixenous genus Leishmania. The L. pyrrhocoris genome (30.4 Mbp in 60 scaffolds) encodes 10,148 genes. Using the L. pyrrhocoris genome, we pinpointed genes gained in Leishmania. Among those genes, 20 genes with unknown function had expression patterns in the Leishmania mexicana life cycle suggesting their involvement in virulence. By combining differential expression data for L. mexicana, L. major and Leptomonas seymouri, we have identified several additional proteins potentially involved in virulence, including SpoU methylase and U3 small nucleolar ribonucleoprotein IMP3. The population genetics of L. pyrrhocoris was also addressed by sequencing thirteen strains of different geographic origin, allowing the identification of 1,318 genes under positive selection. This set of genes was significantly enriched in components of the cytoskeleton and the flagellum.

Figure 1. Schematic representation of Leptomonas pyrrhocoris amino-acid metabolism and some related pathways.

Each dot represents a metabolite and each line represents the presence or absence of a predicted enzyme. Color coding: black, enzyme present; red, enzyme present in most free-living eukaryotic organisms, but lost in L. pyrrhocoris; green, enzyme present in L. pyrrhocoris and Crithidia fasciculata, but absent in Leishmania.

Figure 2. Gene family gains/losses mapped on the tree of kinetoplastids using Dollo parsimony algorithm.

The maximum likelihood tree is based on the alignment of 57 conserved proteins and inferred using the LG + Г + F model and 1,000 bootstrap replicates. Only bootstrap support values lower than 100% are shown. The horizontal bar represents 0.05 substitutions per site. Gene gains dominate at the basal node of trypanosomatids, and at the basal nodes of Leishmaniinae, Leptomonas/Crithidia, American trypanosomes, T. cruzi, and T. brucei. The other internal nodes and leafs are either dominated by losses or have almost equal counts of gains and losses. An inset figure on the right depicts the losses for 99 OGs gained at the Leishmania node. Annotations for proteins within these OGs are shown at the bottom left corner of the inset. Annotations of the known proteins implicated in virulence are in bold.

Figure 3. Approaches used for identification of novel Leishmania virulence factors.

The Venn diagram represents two- and three-way intersections between differential expression datasets (A) (Leptomonas seymouri genes up-regulated at elevated temperature of 35 °C compared to 23 °C), (B) (genes up-regulated in a Leishmania major LV561 virulent isolate compared to an avirulent one), and (C) (differential expression data for L. mexicana developmental stages). Two phylogenetic trees of kinetoplastids with gene family gains/losses mapped using Dollo parsimony algorithm are shown. Gain and loss counts for 20 OGs obtained through overlapping the differential expression datasets are depicted in the tree on the right. The Leishmania cladogram at the bottom shows gains and losses for 21 OGs containing 40 L. mexicana genes gained at the Leishmania node and having differential expression patterns suggesting a potential role in virulence. Leishmania life cycle stages are abbreviated as follows: PRO or P, procyclics; META or M, metacyclics; AMA or A, amastigotes.