Comparative Life Cycle Transcriptomics Revises Leishmania mexicana Genome Annotation and Links a Chromosome Duplication with Parasitism of Vertebrates

Abstract

Leishmania spp. are protozoan parasites that have two principal life cycle stages: the motile promastigote forms that live in the alimentary tract of the sandfly and the amastigote forms, which are adapted to survive and replicate in the harsh conditions of the phagolysosome of mammalian macrophages. Here, we used Illumina sequencing of poly-A selected RNA to characterise and compare the transcriptomes of L. mexicana promastigotes, axenic amastigotes and intracellular amastigotes. These data allowed the production of the first transcriptome evidence-based annotation of gene models for this species, including genome-wide mapping of trans-splice sites and poly-A addition sites. The revised genome annotation encompassed 9,169 protein-coding genes including 936 novel genes as well as modifications to previously existing gene models. Comparative analysis of gene expression across promastigote and amastigote forms revealed that 3,832 genes are differentially expressed between promastigotes and intracellular amastigotes. A large proportion of genes that were downregulated during differentiation to amastigotes were associated with the function of the motile flagellum. In contrast, those genes that were upregulated included cell surface proteins, transporters, peptidases and many uncharacterized genes, including 293 of the 936 novel genes. Genome-wide distribution analysis of the differentially expressed genes revealed that the tetraploid chromosome 30 is highly enriched for genes that were upregulated in amastigotes, providing the first evidence of a link between this whole chromosome duplication event and adaptation to the vertebrate host in this group. Peptide evidence for 42 proteins encoded by novel transcripts supports the idea of an as yet uncharacterised set of small proteins in Leishmania spp. with possible implications for host-pathogen interactions.

Fig 1. Growth history of cells used for RNA extraction.

Data shows growth curve for one of the three replicates. L. mexicana promastigotes (PRO) were maintained in exponential growth by diluting the culture to 1x10⁶ cells ml^-1 every day (blue line). A second promastigote culture was inoculated with 1–2.5x10⁵ cells ml^-1 and left to grow for five days to stationary phase (red line). Stationary phase promastigotes were used to infect bone marrow derived macrophages (BMDM) to produce intracellular amastigotes (AMA) or differentiated to axenic amastigotes (AXA) in Schneider’s medium (SM5.5) for 24h. RNA was extracted from AMA, AXA and PRO on day 7.

Fig 2. Distribution of SLAS, PAS and UTR lengths.

(A) Distribution of assigned SLAS numbers per gene. A SLAS was assigned if at least nine SL-containing reads were mapped to this position across all nine random primed libraries (n = 20,812). (B) Distribution of assigned PAS numbers per gene (n = 95,097). A PAS was assigned if at least six reads terminating in ≥ 5 A were mapped to this position across all six T15VN primed libraries. (C) Distribution of 5’ UTR lengths (without the SL sequence; n = 9,029). (D) Distribution of 3’ UTR lengths (n = 9,029).

Fig 3. Characterisation of novel transcripts.

(A) Size distribution of novel transcripts (n = 936). (B) Size distribution of transcripts derived from genes annotated in TriTrypDB V6 (n = 8,250). (C) Size distribution of largest ORFs found on the sense strand of novel transcripts (n = 936). (D) Size distribution of CDS annotated in TriTrypDB V6 (n = 8,250).

Fig 4. Conservation of novel transcript sequences.

(A) The novel transcripts were used as query sequences in a reciprocal best tblastx search of 12 kinetoplastid genomes. “RBB hits total” indicates the number of reciprocal best tblastx hits returned; “log10 E ≤ -20” indicates the number of hits returned with an E value ≤ 10⁻²⁰ for the reciprocal tblastx search. (B) Venn diagram showing the number of hits returned in a series of reciprocal best tblastx searches comparing the novel transcripts found in L. mexicana (this study), L. major [30] and T. brucei [42].

Fig 5. Conservation of novel transcript sequences across kinetoplastid genomes.

The 936 novel L. mexicana transcripts and 7 control genes were used as queries in tblastx searches of 12 kinetoplastid genomes and the best hits were then used in a reciprocal tblastx search against the complete L. mexicana genome. The heat maps indicate the E value of the returning hits, with darker shades of blue representing lower E values. Sequences that did not return a hit are represented in red. (A) Sequences used as positive controls for conserved CDS (Gene IDs: PFR2, LmxM.16.1430; gGAPDH, LmxM.29.2980; γ-tubulin, LmxM.25.0960; SAS-6, LmxM.34.4280; RPB12, LmxM.20.0490; SmD2, LmxM.32.3190; APX, LmxM.33.0070). (B) Intergenic sequences downstream of the CDS in (A), used as negative controls. (C) Each row represents one of the 936 novel L. mexicana transcripts.

Fig 6. The majority of highly expressed transcripts are shared between AMA, AXA and PRO.

The Venn diagram shows for each cell type the 91 transcripts comprising the top percentile of FPKM values and indicates the extent of overlap between the three datasets.

Fig 7. Differential gene expression between AMA, AXA and PRO.

(A) Table summarising the number of differentially expressed (DE) genes in each pair-wise comparison; “novel” refers to the 936 novel transcripts defined in this study. (B-D) Volcano plots for the comparisons between PRO and AMA (B), PRO and AXA (C), AXA and AMA (D). Each dots represents one transcript; red denotes differential expression (padj ≤ 0.05). Arrows indicate the cell type showing higher expression.

Fig 8. Distribution of differentially expressed genes across chromosomes.

Maps of the 34 L. mexicana chromosomes show the location of genes that are preferentially expressed in AMA (red), PRO (blue) or constitutively expressed (grey).