Spatial relationships of intra-lesion heterogeneity in Mycobacterium tuberculosis microenvironment, replication status, and drug efficacy

Abstract

A hallmark of Mycobacterium tuberculosis (Mtb) infection is the marked heterogeneity that exists, spanning lesion type differences to microenvironment changes as infection progresses. A mechanistic understanding of how this heterogeneity affects Mtb growth and treatment efficacy necessitates single bacterium level studies in the context of intact host tissue architecture; however, such an evaluation has been technically challenging. Here, we exploit fluorescent reporter Mtb strains and the C3HeB/FeJ murine model in an integrated imaging approach to study microenvironment heterogeneity within a single lesion in situ, and analyze how these differences relate to non-uniformity in Mtb replication state, activity, and drug efficacy. We show that the pH and chloride environments differ spatially even within a single caseous necrotic lesion, with increased acidity and chloride levels in the lesion cuff versus core. Strikingly, a higher percentage of Mtb in the lesion core versus cuff were in an actively replicating state, and correspondingly active in transcription/translation. Finally, examination of three first-line anti-tubercular drugs showed that isoniazid efficacy was conspicuously poor against Mtb in the lesion cuff. Our study reveals spatial relationships of intra-lesion heterogeneity, sheds light on important considerations in anti-tubercular treatment strategies, and establishes a foundational framework for Mtb infection heterogeneity analysis at the single bacterium level in situ.

Breathe In, Breathe Out: Metabolic Regulation of Lung Macrophages in Host Defense Against Bacterial Infection

“…Moreover, our current method for lung macrophage extraction may have a substantial impact on metabolism and also lead to a loss of spatial location. Indeed, use of Mtb reporter strains reveals differential replication of bacteria at the core and cuff of lung granulomas ( Lavin and Tan, 2022 ), which demonstrates the importance of considering spatial relationships of host-bacteria interactions and the possibility of using bacterial reporters to probe lung macrophage metabolism in situ ( Macgilvary and Tan, 2018 ). Finally, one of the most important perspectives is to translate our discoveries into human treatments.…”

PrrA modulates Mycobacterium tuberculosis response to multiple environmental cues and is critically regulated by serine/threonine protein kinases

“…To directly examine this, we tested if PrrA is essential for Mtb growth within macrophages and in a murine model of Mtb infection. Knockdown of prrA in murine infections with the parental PrrA-DUC/ ΔprrA Mtb was accomplished via the administration of doxycycline in the drinking water, which robustly allows control of tetracycline-based inducible systems in Mtb while having no effect on bacterial load in this model, as previously established [11,58,59]. As expected given the essentially of prrA, knockdown of prrA significantly attenuated Mtb growth both within macrophages and mice (Fig Most strikingly, the PrrA-T6A variant was significantly attenuated in its ability to colonize a…”

“…The ability of STPKs to phosphorylate multiple targets (tens to hundreds) mark them as systems with immense capacity as masters of integrating and layering regulation. However, while STPKs (11) and TCSs ( 12) are both well-represented in the Mtb genome, much remains unknown about how these two vital environmental response systems may work together, with just three examples to our knowledge exploring this interplay biochemically [35,36,65]. The identity of the STPKs that regulate PrrA is currently unclear.…”

“…Similarly, Mtb response to NO and hypoxia is intimately linked, with extensive overlap in the bacterial transcriptional response to these two signals, and the DosRS(T) two-component system (TCS) regulating a core subset of genes in response to either signal [2,[4][5][6][7]. These environmental signals change in predictable manners within the host, reflecting both location and the status of the immune response [1,3,[8][9][10][11]. They further affect Mtb growth status, with Mtb growth slowed at acidic pH [12,13], and the bacteria entering an adaptive state of growth arrest upon extended exposure to NO or hypoxia [2,14,15].…”

“…While the presence of STPKs in bacterial genomes is increasingly appreciated, only one or two STPKs/bacterial genome have generally been predicted versus the larger number of TCSs [30][31][32][33]. In contrast, STPKs are prominent in Mtb, with an almost equivalent number of STPKs (11) present as TCSs (12) [30]. Although hundreds of proteins are predicted to be phosphorylated by STPKs in Mtb, the role of STPKs in Mtb environmental sensing/response remains poorly understood.…”