Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions

Abstract

Background: Parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate.

Results: We performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure.

Conclusions: The gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.

Fig. 1

Characterization of L. major intracellular growth and proportion of RNA from the parasite. Mouse macrophages infected with L. major were collected at 4, 24, 48, and 72 hpi and subjected to transcriptional profiling by RNA-seq. An average of 87 % of macrophages were infected across all samples. Bar plots are used to illustrate a the average number of parasites observed per 100 host cells and b the average percentage of trimmed RNA-seq reads that map to the L. major genome. Standard errors bars are shown. No statistically significant changes were observed between timepoints

Fig. 2

Global gene expression profiles of L. major parasites and their murine macrophage host cells. RNA-seq was carried out on mouse macrophages infected with L. major at 4, 24, 48, and 72 hpi as well as on the metacyclic promastigotes used for the infection. Principal component analysis (PCA) plots and heatmaps of hierarchical clustering analyses using Euclidean distance are shown for the L. major (a, c) and mouse (b, d) transcriptomes over the course of the experiment. The analyses were performed using all annotated protein-coding genes following filtering for low counts and quantile normalization after accounting for batch effects in the statistical model (8479 genes for L. major and 12552 genes for mouse). In the PCA plots, the first two principal components are shown on the X and Y axes, respectively, with the proportion of total variance attributable to that PC indicated. Each experimental sample is represented as a single point with color indicating sample type/timepoint and shape indicating experimental batch. Colors along the tops of the heatmaps indicate the sample type/timepoint and colors along the left sides of the heatmaps indicate the experimental batch. Samples are named according to sample type (“metac” for L. major metacyclic promastigotes, “amast” for L. major amastigotes, “uninf” for uninfected mouse macrophage, or “inf” for L. major-infected mouse macrophage), timepoint (4, 24, 48, or 72 hpi) and experimental batch (a, b, or c) (see Additional file 1)

Fig. 3

Differentially expressed genes in L. major parasites and their murine macrophage host cells. RNA-seq was carried out on mouse macrophages infected with L. major at 4, 24, 48, and 72 hpi as well as on the metacyclic promastigotes used for the infection. Pairwise comparisons were done to determine differentially expressed (DE) genes from uninfected vs. infected mouse samples at each timepoint (a, top) and between timepoints (a, middle), and for L. major parasite samples between timepoints (a, bottom). Box length depicts the number of DE genes either downregulated (left) or upregulated (right) at an adjusted P value of < 0.05 with the total number of down- and upregulated genes shown. Color hue indicates sample type/timepoint as defined in Fig. 2 and color shade indicates the proportion of genes with > 4-fold differential expression (dark), between 2- and 4-fold differential expression (medium), or 2-fold differential expression (light). The DE gene lists for uninfected vs. infected mouse cells at each timepoint were compared and the overlap shown as a Venn diagram in (b). The complete lists of DE genes are provided in Additional file 3 for mouse and Additional file 6 for L. major