Transcriptomic profiling of Leishmania parasites and host macrophages during an infection

Abstract

Leishmania parasites cause leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The parasite's life cycle is divided between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must avoid being killed by the innate and adaptive immune responses. We performed transcriptomic profiling using RNA-seq to simultaneously identify global changes in gene expression in Leishmania parasites across multiple lifecycle stages and in infected macrophages from both murine and human hosts. Using a novel approach based on a dual statistical test to identify genes that were differentially expressed relative to both uninfected macrophages and macrophages that had ingested inert particles, we were able to filter out genes that were differentially regulated as part of a general phagocytic response and thereby select genes specific to Leishmania infection. The most substantial and dynamic Leishmania-specific differential expression responses were observed during early infection, while changes observed later were common to phagocytosis more generally. An evaluation of RNA processing events within the parasite revealed precise UTR boundaries for a majority of genes and widespread alternative trans-splicing and polyadenylation. Collection of data from multiple biological replicates, the use of matched host control samples, careful statistical analysis of variation, and removal of batch effects enabled the detection of biological differences between samples and timepoints with high confidence and sensitivity. Pathway and gene ontology analyses provided insights into the higher level processes activated across parasite developmental stages and during intracellular infection to reveal signatures of Leishmania differentiation and infection.