Fig 1.
Effect of treatment in axenic culture.
(a) Timeline of treatment for experimental arms for in vitro study. Cultures were untreated, treated for 6 hours, or treated for 48 hours. Cultures underwent washout to remove drugs, diluted to an OD of 0.05, then allowed to grow until exceeding an OD600 of 2 or 15 days of recovery. (b) CFU burden after 0, 6, and 48 hours of treatment. Each dot represents one sample. The line connects the median values. (c) RS ratio as displayed in (b). (d-e) Volcano plot showing log2 fold changes and −log10 P-values of genes differentially expressed after (d) 6-hour or (e) 48-hour treatment compared to pre-treatment control. Genes significantly down- and up-regulated relative to control (adj. P < 0.05) are shown in blue and red, respectively. (f) Log fold change of genes after 48-hour treatment compared to pre-treatment control versus 6-hour treatment compared to pre-treatment control. Purple shading indicates genes with concordant fold-change direction and significance between treatment times. Teal shading indicates genes that were significantly differentially expressed under one treatment duration but not both. Gold shading indicates genes that were significant for both treatment durations but in opposite directions. Gray shading indicates genes that were not significantly differentially expressed in either study. Table specifies the number of genes per category. (g) Hierarchical clustering of genes differentially expressed over time with HRZE treatment in axenic culture (N = 3,135 genes). The heatmap shows the batch-adjusted, VST-normalized, scaled gene expression averaged across samples. Clustering identified four broad patterns. Dot plots show the average expression for each cluster across time points. Each point represents an individual sample. Horizontal lines indicate average values. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treatment, respectively. (h-i) Dot plots indicate Mtb gene categories that were enriched in (h) clusters 1 and 2 or (i) clusters 3 and 4. Dot size indicates the number of genes from a category that were present in the clusters and x-axis indicates what percent of the category was present. Coloration indicates significance. Only the top 10 enrichments are shown.
Fig 2.
Overview of recovery in axenic culture.
(a) OD600, (b) change in CFU, and (c) RS ratio of samples during the PAE phase of recovery after being diluted to OD600 of 0.05 and allowed to grow in the absence of HRZE. Each dot represents one sample, and lines connect the median values across timepoints for untreated control (green), 6-hour treatment (orange), and 48-hour treatment (purple) group. (b) Timeline for sampling each in vitro treatment group for SEARCH-TB sequencing and analysis. (e) Hierarchical clustering of genes differentially expressed during PAE phase (N = 3,507 genes). The heatmap shows the batch-adjusted, VST-normalized, scaled gene expression averaged across samples. Clustering identified four broad patterns. Dot plots show the average expression for each cluster across time points. Each point represents an individual sample from 6-hour treatment (orange) or 48-hour treatment (purple) groups. Horizontal lines indicate average values. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treatment, respectively.
Fig 3.
Transcriptional recovery in axenic culture.
(a) The heatmap shows the batch-adjusted, VST-normalized, scaled gene expression averaged across samples for specified groups of gene categories related to metabolism. (b-d) Average of batch adjusted, VST-normalized, scaled gene expression in each treatment group over time for genes involved in (b) cytochrome bcc/aa3 supercomplex, (c) cytochrome bd oxidase, and (d) protein translation and modification. Each point represents an individual sample, and the lines connect the mean for each time point. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treated, respectively. (e) Heatmap as in (a) for gene categories relevant to cell wall synthesis and maintenance. (f-g) Average expression plots as in (b) for (f) chaperone heat shock proteins and (g) the DosR regulon. (h) Heatmap as in (a) for sigma factors A-M. (i) Hierarchical clustering of toxin genes (N = 73 genes) identified three broad patterns. Dot plots show the average expression for each cluster across time points. Each point represents an individual sample from pre-treatment control (black), 6-hour treatment (orange), or 48-hour treatment (purple). Horizontal lines indicate average values. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treatment, respectively. (j-l) Average expression plots as in (b) for (j) ESX1, (k) ESX3, (l) ESX4, (m) DNA gyrases, (n) the clpP1P2 complex, and (o) oxidative stress.
Fig 4.
Effect of treatment in murine samples.
(a) Timeline of mouse infection, treatment and PAE phase for experimental arms of murine study. Mice were untreated (preRx), treated for 2 weeks, or treated for 4 weeks. For treatment arms, treatment cessation was followed by a 28-day PAE phase. (b) CFU burden after 0, 2, and 4 weeks of treatment. Each dot represents one mouse sample. The line connects the median values. (c) RS ratio as displayed in (b). (d) Log fold change of genes after 4-week treatment compared to pre-treatment control versus 2-week treatment compared to pre-treatment control. Purple shading indicates genes with concordant fold-change direction and significance between treatment times. Teal shading indicates genes that were significantly differentially expressed under one treatment duration but not both. Gold shading indicates genes that were significant for both treatment durations but in opposite directions. Gray shading indicates genes that were not significantly differentially expressed in either study. Table specifies the number of genes per category. (e) Hierarchical clustering of genes differentially expressed over time with HRZE treatment in murine samples (N = 2,724 samples). The heatmap shows the batch-adjusted, VST-normalized, scaled gene expression averaged across samples. Clustering identified three broad patterns. Dot plots show the average expression for each cluster across time. Each dot represents an individual sample. Horizontal lines indicate average values. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treatment, respectively.
Fig 5.
Overview of recovery in murine samples.
(a) Change in CFU and (b) RS ratio of samples during the PAE phase of recovery. Each dot represents one sample, and lines connect the median values across timepoints for 2-week treatment (blue) and 4-week treatment (pink) groups. (c) Hierarchical clustering of genes differentially expressed over PAE phase (N = 3,017 genes). The heatmap shows the batch-adjusted, VST-normalized, scaled gene expression averaged across samples. Clustering identified four broad patterns. Dot plots show the average expression for each cluster across time points. Each point represents an individual sample from 2-week treatment (blue) or 4-week treatment (pink) group. Horizontal lines indicate average values. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treatment, respectively.
Fig 6.
Transcriptional recovery in murine samples.
(a) The heatmap shows the batch-adjusted, VST-normalized, scaled gene expression averaged across samples for specified groups of gene categories related to metabolism. (b-d) Average of batch adjusted, VST-normalized, scaled gene expression in each treatment group over time for genes involved in (b) cytochrome bcc/aa3 supercomplex, (c) cytochrome bd oxidase, and (d) protein translation and modification. Each point represents an individual sample, and the lines connect the mean for each time point. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treated, respectively. (e) Heatmap as in (a) for gene categories relevant to cell wall synthesis and maintenance. (f-g) Average expression plots as in (b) for (f) chaperone heat shock proteins and (g) the DosR regulon. (h) Heatmap as in (a) for sigma factors A-M. (i) Hierarchical clustering of toxin genes (N = 73 genes) identified three broad patterns. Dot plots show the average expression for each cluster across time points. Each point represents an individual sample from 2-week treatment (blue) or 4-week treatment (pink). Horizontal lines indicate average values. Values are centered around the average value for the pre-treated samples so that points above and below zero represent upregulation and downregulation relative to pre-treatment, respectively. (j-l) Average expression plots as in (b) for (j) ESX1, (k) ESX3, (l) ESX4, (m) DNA gyrases, (n) the clpP1P2 complex, and (o) oxidative stress.