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

Abstract

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.

Keywords: Mycobacterium; Mycobacterium tuberculosis; gene expression; tolerance.

Fig 1

Visual summary of methods previously used to quantify the Mtb transcriptome in vivo. Each horizontal arrow represents a distinct combination of enrichment and quantification. Varying enrichment methods are represented via the symbols shown in the key. SEARCH-TB is a unique combination of enrichment (eukaryotic cell lysis + targeted amplification) followed by quantification via RNA-seq that has enabled transcriptome evaluation in mice treated for weeks with a potent combination regimen. Image created with Biorender.com.

Fig 2

Evaluation of SEARCH-TB platform. (a and b) Volcano plot showing log₂ fold changes and −log₁₀ P-values induced by 24 h in vitro INH exposure as quantified by RNA-seq (a) and SEARCH-TB (b). Genes significantly down- and up-regulated with INH exposure relative to control (adj. P < 0.05) are shown in blue and red, respectively. (c) Comparison of differential expression between INH-treated samples and control samples from RNA-seq or SEARCH-TB data. Purple shading indicates genes with concordant fold-change direction and significance between RNA-seq and SEARCH-TB. Green shading indicates genes that were significant in RNA-seq or SEARCH-TB results but not both. Gold shading indicates genes that were significant for both RNA-seq and SEARCH-TB but in opposite directions. Gray shading indicates genes that were not significantly differentially expressed in either RNA-seq or SEARCH-TB. (d) Comparison of INH versus control fold changes from RNA-seq data versus SEARCH-TB data. Purple, green, gold, and gray colors have the same meaning as in (c).

Fig 3

Overview of transcriptional response to HRZE in mice and in vitro experiments. (a) The first two principal components of gene expression for mouse and in vitro samples. In vitro samples are colored using shades of yellow while mouse samples are colored using shades of green. Time points are differentiated by the shade and shape of each point. (b) Volcano plot summarizing the differential expression between Mtb in mice and in vitro prior to treatment. Genes significantly down- (blue) or up-regulated (red) in mice relative to in vitro (adj. P < 0.05) are shown. (c and d) Mtb CFU and RS ratio values for (c) mouse samples at the pre-treatment control time point and 14 and 28 days after HRZE treatment initiation, and (d) in vitro samples at the pre-treatment control time point and 4 and 8 days after HRZE treatment initiation. (e and f) Volcano plots summarizing the differential expression between Mtb in (e) 14 days HRZE-treated and control mouse samples and (f) 28 days HRZE-treated and control mouse samples. (g) Estimated gene expression over time in mice for genes significantly differentially expressed between at least two treatment time points (N = 2,429). Values are row-scaled, with red and blue indicating higher and lower expression, respectively. Hierarchical clustering of genes identified four broad patterns. (h) Average log₂ fold changes for each of the four clusters relative to control. Values above and below zero represent up- and down-regulation relative to control, respectively. (i) Comparison of differential expression between mouse (day 28) or in vitro (day 8) relative to respective controls. Purple shading indicates genes with concordant fold-change direction and significance between mouse and in vitro experiments. Green shading indicates genes that were significant for either mouse or in vitro experiments but not both. Gold shading indicates genes that were significant for both mouse and in vitro experiments but in opposite directions. Gray shading indicates the genes that were not differentially expressed with HRZE treatment either in mouse or in vitro experiments. (j) Comparison of fold changes between mouse (day 28) or in vitro (day 8) relative to respective controls. Purple, green, gold, and gray colors have the same meaning as in (i).

Fig 4

Summary of gene set enrichment and transcriptional changes in biological processes. (a) Gene categories significantly enriched for genes differentially expressed between day 28 and control murine samples. The percentage of genes in each category significantly up- (red) or down-regulated (blue) for each comparison is illustrated. Asterisks indicate statistical significance (adj. P < 0.05). (b) Fold change of ribosomal protein genes in mice on day 28 (left) and in vitro (right) on day 8, relative to control. Red bars indicate the four alternative C-ribosomal protein paralogs. (c and e) Fold-change values in mice at days 14 and 28 (left) and in vitro (right) at days 4 and 8, relative to control for (c) FAS-II and (e) antigen 85 gene sets. (d) Graphical representation of changes in the peptidoglycan synthesis, modification, and recycling pathways in mice on day 28 relative to control. Log₂ fold-change values for genes in the process are indicated by the color of each box and the thick outlines of boxes represent genes that are significantly differentially expressed. Figure adapted from Maitra et al., 2019 (45). Image created with Biorender.com. (f) Fold change of ESX-1 genes in mice on day 28 (left) and in vitro (right) on day 8, relative to control. Red bar represents esxA and blue bar represents esxB.

Fig 5

Summary of transcriptional changes in biological processes. Fold change in mice (left) and in vitro (right), relative to control for (a) ATP synthetase, (b) cytochrome bcc/aa3 supercomplex and the bd oxidase, (c) glyoxylate bypass, (d) heat shock proteins (red bar represents hspX), (e) dosR, (f) the Rv2686c-2688c efflux pump, and (g) DrrABC efflux pump gene sets.