Dual RNA-Seq analysis of Mus musculus and Leishmania donovani transcriptomes

Parasitic protozoa of the genus Leishmania cause a spectrum of disease, affecting 12 million people worldwide. This project aimed to investigate the effect of Leishmania donovani infection on the gene expression of healthy/WT (Black 6) and immunocompromised (RAG2KO) mice. Differences in the gene expression of parasites in inoculum and tissue were also elucidated.

WT and RAG mice were infected using an L. donovani inoculum, and were euthanised after 28 days. Harvested spleens (and the inoculum) were used to generate RNA samples, from which mRNA was isolated and purified. Transcriptome data was generated using dual RNA-Seq approaches from the mRNA samples. After appropriate pre-processing, data underwent a number of bioinformatic analyses to explore differential gene expression, such as Gene Ontology, Gene Set Enrichment, and KEGG Pathway analysis.

Comparison of different mouse spleen transcriptomes revealed that even in uninfected mice, WT mice more highly express genes related to immunoglobulins when compared with their immunocompromised counterparts. Healthy mice were found to react to infection through the induction of inflammatory response, and the production of NOX generating species. RAG mice still upregulated immunoglobulin-related genes in response to infection, despite their inability to generate antibodies, T-cells, and B-cells. However, RAG modulation of haeme and iron metabolism may contribute to defence against the parasites despite a lack of acquired immunity.

Differences in the amastin, the key glycoprotein on the surface on intracellular-stage parasites, are apparent between the inoculum and tissue parasites, which may reflect microenvironment adaptation. Additionally, tissue-derived parasites showed significant upregulation of genes related to gene expression control, such as histones and DNA-packing.

These experiments are among the first attempts to in vivo transcriptome sequence mice and Leishmania simultaneously, a powerful approach giving insight to action and reaction. However, these techniques are not without challenge, such as low parasite read counts.