Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing

The human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio. We infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette-Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism. Dual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection.