Abstract
Mycobacterium bovis, the agent of bovine tuberculosis, causes an estimated $3 billion annual losses to global agriculture due, in part, to the limitations of current diagnostics. Development of next-generation diagnostics requires a greater understanding of the interaction between the pathogen and the bovine host. Therefore, to explore the early response of the alveolar macrophage to infection, we report the first application of RNA-sequencing to define, in exquisite detail, the transcriptomes of M. bovis-infected and non-infected alveolar macrophages from ten calves at 2, 6, 24 and 48 hours post-infection. Differentially expressed sense genes were detected at these time points that revealed enrichment of innate immune signalling functions, and transcriptional suppression of host defence mechanisms (e.g., lysosome maturation). We also detected differentially expressed natural antisense transcripts, which may play a role in subverting innate immune mechanisms following infection. Furthermore, we report differential expression of novel bovine genes, some of which have immune-related functions based on orthology with human proteins. This is the first in-depth transcriptomics investigation of the alveolar macrophage response to the early stages of M. bovis infection and reveals complex patterns of gene expression and regulation that underlie the immunomodulatory mechanisms used by M. bovis to evade host defence mechanisms.
Highlights
M. bovis is primarily transmitted via inhalation of aerosolized bacteria, with the primary site of infection being the lung[6]
The pathogen is phagocytosed by host alveolar macrophages, which serve as key effector innate immune cells and usually can kill intracellular bacilli or contain infection via the activity of inflammatory cytokines[7]
We hypothesised that mechanisms used by pathogenic mycobacteria to overcome innate immunity and establish infection would be revealed through analysis of the gene expression changes in the macrophage response to infection
Summary
The laboratory methods have previously been described by us[25] and a summary is provided below. The samples used for RNA-seq library preparation comprised M. bovis-infected and non-infected samples from each post-infection time points across 10 animals (except for one animal that did not yield sufficient macrophages for the 48 hpi time point). Uniquely aligned paired-end reads were used to obtain raw counts for all bovine genes (B. taurus UMD3.1.71 Ensembl genome annotation30) based on sense strand data with the featureCounts software[31]. The uniquely aligned paired-end reads not assigned to any sense or antisense gene were used to compute raw counts for novel genes using the featureCounts software and the de novo B. taurus genome annotation. Two-tailed paired sample t-tests and pairwise Wilcoxon signed-rank tests were used to assess statistically significant gene expression fold-changes for normally and non-normally distributed data, respectively. The Pearson correlation (r) between RT-qPCR and RNA-seq gene expression fold-change was estimated
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