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

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. Author summaryA critical aspect of tuberculosis disease, caused by the bacterium Mycobacterium tuberculosis (Mtb), is non-uniformity in bacterial physiology and disease progression. This heterogeneity is thought to be a major reason for the need for prolonged treatment, and understanding what and how facets of the infection differ within a host or single lesion is thus vital for the development of improved therapeutic strategies. However, elucidation and direct demonstration of bacterial non-uniformity during whole animal infection, in the context of intact tissue architecture, has been extremely challenging and consequently lacking. We show here using an integrated imaging approach that Mtb experiences different pH and chloride microenvironments depending on its sublocation within a structured lesion, which strikingly correlates with Mtb replication status and transcriptional/translational activity. We further find that these spatial differences impact critically on drug efficacy, with the first-line anti-tubercular drug isoniazid demonstrating conspicuously poor efficacy against Mtb residing in the structured lesion cuff, versus the bacteria present in the lesion core. Our study reveals how several aspects of non-uniformity in Mtb infection biology are related to spatial location within a structured lesion, and lays the groundwork for analysis of Mtb infection heterogeneity at the single bacterium level in intact tissue.

Automated summary

This study utilized an integrated imaging approach to investigate the microenvironment heterogeneity of Mycobacterium tuberculosis within a single lesion, revealing spatial differences in pH and chloride environments that impact the replication status of Mtb and drug efficacy.