Direct bacterial transcript visualization illuminates spatiotemporal environmental adaptation of Mycobacterium tuberculosis in the lung.

Spatiotemporal environmental variation results in marked heterogeneity in bacterial infection progression and disease outcome, with vital consequences for treatment success. For the globally important pathogen Mycobacterium tuberculosis (Mtb), while the pronounced intra-host spatial heterogeneity in lesion immune cell composition and phenotype has been well-described, the highly complex Mtb cell envelope has presented a particular challenge for the required equivalent insight into bacterial heterogeneity. Here, we develop hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH)-based methodology for Mtb mRNA visualization in the context of intact lung and lesion architecture. In combination with an Mtb transcriptional/translational activity reporter, we reveal spatiotemporal differences in gene expression relating to Mtb lipid metabolism, response to key environmental signals, and the ESX-1 type VII secretion system. Our results establish a framework for in situ analysis of Mtb mRNA, opening the path to elucidating critical bacterial drivers that underlie the marked heterogeneity in Mtb-host interactions.