Figure 2. Identification of an Mtb In Vivo Signature in Mice.

(A) Principal-component analysis (PCA) of the Mtb transcriptome from three different environmental conditions: in vivo, cell culture (BMDM), and broth (7H9-OADC).

(B) Heatmap showing relative expression levels for the “in vivo signature,” a set of 180 genes upregulated only during growth in lung macrophages.

(C) Functional characterization of the in vivo gene signature shows how many of the genes belong to the “conserved hypothetical” class with undefined function.

(D) Protein-protein network analysis of the “in vivo signature” reveals a cluster of genes involved in cholesterol degradation (Kstr2), response to nitrosative and oxidative stresses (ergothioneine), and nitrogen assimilation and export machinery (Esx-5). Only high-confidence (>0.7) interactions were used to build the network.

(E) Heatmap showing relative expression levels for the genes in the cholesterol degradation pathway Kstr2.

(F) Boxplot of the main genes related to the MMP (MutA, MutB) and MCC (PrpC, PrpD) pathways. The transcriptional profile of Mtb from in vivo samples shows upregulation of the genes related to the MMP pathway but not of those involved in the recycling of the propionyl-CoA pool through the MCC.

(G and H) Heatmaps showing relative expression levels for genes in the inorganic nitrogen (G) and ESX-5 (H) pathways in Mtb.

(I) Violin plots showing expression levels (in log-normalized counts) of genes involved in the ergothioneine biosynthesis pathway.

Where appropriate, for all plots the statistical significance is shown on the picture (*adjusted p value [p-adj.] < 0.05, **p-adj. < 0.01, ***p-adj. < 0.001, and ****p-adj. < 0.0001). q values for comparisons among the groups were calculated using the Wald test as implemented in the DESeq2 pipeline.