Transcriptional adaptation of
Mycobacterium tuberculosis
that survives prolonged multi-drug treatment in mice
Authors: Wynn Elizabeth A. , Dide-Agossou Christian , Reichlen Matthew , Rossmassler Karen , Al Mubarak Reem , Reid Justin J. , Tabor Samuel T. , Born Sarah E. M. , Ransom Monica R. , Davidson Rebecca M. , Walton Kendra N. , Benoit Jeanne B. , Hoppers Amanda , Loy Dorothy E. , Bauman Allison A. , Massoudi Lisa M. , Dolganov Gregory , Strong Michael , Nahid Payam , Voskuil Martin I. , Robertson Gregory T. , Moore Camille M. , Walter Nicholas D. 
Published: October 2023

ABSTRACT

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.