Fig. 2. Single-bacillus RS ratio in situ indicates that Mtb rRNA synthesis is lower in caseum than the granuloma’s inflammatory rim.

a H&E-stained section of a single typical lung granuloma from C3HeB/FeJ mouse. Most of the lesion is comprised of caseous necrosis (1). The caseum is surrounded by a rim of inflammatory cells that include viable and degenerate neutrophils and heavily vacuolated macrophages (2). The granuloma is contained by an outer rim of compressed lung tissue, fibrosis, and infiltrating leukocytes (3). b Granuloma from C3HeB/FeJ mouse lung with multiplexed ISH overlay staining for 23S rRNA (green), pre-rRNA (red), and DAPI for host-cell nuclei (blue). The channels for ISH are shown individually in c and d. c 23S rRNA ISH identified Mtb present throughout the granuloma. d pre-rRNA ISH indicated lower Mtb rRNA synthesis in the caseum compared with the inflammatory rim. e Graphical analysis as well as statistical testing of the RS ratio by ISH showed that there was greater population heterogeneity in rRNA synthesis in the inflammatory rim (orange) than in the caseum (blue). Components of this raincloud plot are: (1) density plots for the distribution of 164,878 RS ratio values for individual bacilli in a single granuloma on a log₁₀ scale, (2) scatterplots to visualize all points measured, and (3) boxplots to present the range of values in the RS ratio. The center and ends of the box represent the median and first and third quartiles of the RS ratio. The boxplot whiskers represent the maximum and minimum values in each group. f Magnification of high-powered images depicts co-occurrence of rRNA signals within individual bacilli. g Further magnification demonstrates 23S signals distributed in a reticular pattern around a central confluence of pre-rRNA signals. Panels a–d were imaged at ×40; scale bars represent 500 µm. Z-stacked images surrounding this image are provided in Supplementary Fig 4. Panels f and g were imaged at ×63; scale bars represent 10 and 5 µm, respectively. Panels a–g are results from a single lung granuloma. Replicate results from two additional granulomas from two different mice in separate independent experiments are provided in Supplementary Fig. 2.