Transcriptional adaptation of Mycobacterium tuberculosis that survives prolonged multi-drug treatment in mice.

To address the ongoing global tuberculosis crisis, there is a need for shorter, more effective treatments. A major reason why tuberculosis requires prolonged treatment is that, following a short initial phase of rapid killing, the residual Mycobacterium tuberculosis withstands drug killing. Because existing methods lack sensitivity to quantify low-abundance mycobacterial RNA in drug-treated animals, cellular adaptations of drug-exposed bacterial phenotypes in vivo remain poorly understood. Here, we used a novel RNA-seq method called SEARCH-TB to elucidate the Mycobacterium tuberculosis transcriptome in mice treated for up to 28 days with standard doses of isoniazid, rifampin, pyrazinamide, and ethambutol. We compared murine results with in vitro SEARCH-TB results during exposure to the same regimen. Treatment suppressed genes associated with growth, transcription, translation, synthesis of rRNA proteins, and immunogenic secretory peptides. Bacteria that survived prolonged treatment appeared to transition from ATP-maximizing respiration toward lower-efficiency pathways and showed modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pump expression. Although the pre-treatment in vivo and in vitro transcriptomes differed profoundly, genes differentially expressed following treatment in vivo and in vitro were similar, with differences likely attributable to immunity and drug pharmacokinetics in mice. These results reveal cellular adaptations of Mycobacterium tuberculosis that withstand prolonged drug exposure in vivo, demonstrating proof of concept that SEARCH-TB is a highly granular pharmacodynamic readout. The surprising finding that differential expression is concordant in vivo and in vitro suggests that insights from transcriptional analyses in vitro may translate to the mouse. IMPORTANCE A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of Mycobacterium tuberculosis that survive drug exposure in vivo have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated Mycobacterium tuberculosis phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen in vitro. This first view of the transcriptome of the minority Mycobacterium tuberculosis population that withstands treatment in vivo reveals adaptation of a broad range of cellular processes, including a shift in metabolism and cell wall modification. Surprisingly, the change in gene expression induced by treatment in vivo and in vitro was largely similar. This apparent "portability" from in vitro to the mouse provides important new context for in vitro transcriptional analyses that may support early preclinical drug evaluation.