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

Anna-Lisa E Lawrence; Shumin Tan

doi:10.1126/sciadv.aeb8377

. 2026 Aug 12;12(33):eaeb8377. doi: 10.1126/sciadv.aeb8377

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

Anna-Lisa E Lawrence ¹, Shumin Tan ^1,^*

PMCID: PMC13464648 PMID: 42585320

Abstract

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.

INTRODUCTION

Bacterial adaptation to changes in the local environment is a critical driver of infection and disease outcome, with disruption of the bacterium’s ability to adapt and overcome host-mediated stressors resulting in colonization failure (1–4). In addition to global changes in the host environment directed by factors such as the onset of adaptive immunity with time (5, 6), there has been a burgeoning appreciation for the spatial differences that also exist, facilitated by the development of techniques such as spatial transcriptomics, iterative protein or in situ hybridization-based microscopy, and imaging mass spectrometry-based methods (7–13). This encompasses spatial differences ranging from host cell types and immune responses within lung or skin lesions during Mycobacterium tuberculosis (Mtb) or Mycobacterium leprae infection respectively (14–18), to neutrophil infiltration and host cell metabolic changes in different regions of the cornea during Pseudomonas aeruginosa infection (19), and to differences in availability of vital metals within the kidney during Staphylococcus aureus infection (13). Such spatial heterogeneity in host cellular composition and response dictate a need for equivalent understanding of how the response and physiology of the infecting bacteria vary in tissue context, to elucidate specific bacterial niche adaptation responses and enable effective targeting of the bacteria for disease resolution.

To this end, fluorescent bacterial reporter strains have provided intriguing first insight into how bacterial gene expression changes at the single bacterium level during in vivo infection in spatial context. This spans studies on Vibrio cholerae toxin-related gene expression during intestinal infection (20), the response of Salmonella and Yersinia pseudotuberculosis to nitric oxide stress in the spleen (21, 22), and intra-lesion differences in Mtb exposure to acidic pH and high chloride levels (23). For Mtb, our studies with reporter strains have further revealed the correlation between a more acidic pH/higher chloride environment with decreased bacterial replication and transcriptional/translational activity, with a corresponding decrease in efficacy of drugs that target actively growing bacteria against Mtb residing in the lesion sublocation with more acidic pH/higher chloride (23). Fluorescent reporters have thus been invaluable in uncovering key spatial changes in bacterial physiology during infection, but carry limitations such as restrictions in time points, in cases where the reporters are encoded on episomal plasmids, and in throughput. New methods that excitingly seek to enable more global probing of spatial changes in bacterial gene expression at the single bacterium level have more recently been reported, including the development of parallel and sequential FISH (par-seq FISH), which allowed for the detection of 105 different P. aeruginosa genes in planktonic and biofilm cultures (24), and multiplexed error robust fluorescence in situ hybridization (MER-FISH), which coupled expansion microscopy with FISH-based labeling to detect individual bacterial mRNA in broth-grown Escherichia coli, and in Bacteroides thetaiotaomicron during colon colonization in spatial context (25). Application of FISH-based techniques to the analysis of mRNA from bacteria with highly complex cell envelopes or thick peptidoglycan layers, which resist standard permeabilization methods and substantially impede probe access, however, presents an additional technical hurdle. In this regard, Mtb, the causative agent of tuberculosis that remains the leading global cause of death from an infectious disease (26), represents a particular challenge, with an outer capsule-like layer, mycolic acids, and arabinogalactan, peptidoglycan, and lipoarabinomannan layers beyond the plasma membrane (27). At the same time, extensive studies revealing the spatial heterogeneity of host cellular architecture and response in hallmark Mtb lesions (granulomas) in the past few years (15–18) emphasize the critical need to understand how Mtb response differs in spatial lesion context.

Here, we report the development of a method that enables direct, quantitative detection of individual Mtb operons via hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) (7, 11), at single bacterium resolution in the context of intact tissue and lesion architecture. We focus on three key facets of Mtb host infection (lipid utilization, nitric oxide (NO)/hypoxia exposure, and type VII secretion system function) to establish this method in our foundational study, utilizing the C3HeB/FeJ murine model of Mtb infection that recapitulates the hallmark necrotic lesion type found during human infection (28, 29). Our results reveal not just a temporal upregulation of Mtb lipid-responsive genes as infection progresses, but spatial differences in expression of these genes between Mtb residing in the macrophage-dominant cuff versus in the neutrophil-dominant core edge of necrotic lesions. Expression of the hspX operon that responds very robustly to initial exposure to NO and hypoxia showed striking differences in Mtb present in the lesion cuff versus in the core center, with the highest levels observed in the core edge of necrotic lesions. Finally, spatiotemporal differences in expression of the espACD ESX-1 type VII secretion system substrates were also observed, with highest expression in Mtb residing in the cuff of necrotic lesions and in non-necrotic macrophage-rich lesions, and a trend towards reduced expression at the latest 16-week timepoint. Together with results from a chromosomally-encoded Mtb transcriptional/translational activity reporter, our work provides crucial insight into spatiotemporal changes in Mtb physiology and response during host infection, and raise intriguing concepts for future study. By establishing a framework and HCR-FISH methodology for analysis of Mtb mRNA, our study further opens the path to incorporating understanding of the bacterial aspect in elucidating factors that underlie the marked heterogeneity in Mtb-host interactions, enabling the holistic comprehension required to account for the critical impact of heterogeneity on infection outcome in designing effective therapeutic strategies.

RESULTS

Mtb transcriptional/translational activity within necrotic lesions decreases as infection progresses

Mtb growth state strongly impacts infection outcome and treatment efficacy (23, 30–33), yet spatiotemporal changes in Mtb replication in the context of necrotic lesion formation and maturation is not well understood. To address this question, we utilized a doxycycline-inducible monomeric Kusabira Orange (mKO) reporter Mtb strain that also carries a constitutively expressed mCherry (P₆₀₆’::mKO-tetON, smyc’::mCherry), both encoded on the chromosome (23, 34). Active Mtb transcribe and translate mKO upon exposure of the mice to doxycycline inducer, enabling spatial analysis of which bacteria are active in different lesion sublocations, and we have shown that signal from this reporter tightly correlates with bacterial replication (23). While we had previously found that penetration of doxycycline into the center of the lesion core (“core center”; Fig. 1A) appears impeded as no mKO signal is observed, distinct differences between mKO signal induced upon doxycycline administration is observed between Mtb present in the lesion cuff versus in the edge of the lesion core (“core edge”) (Fig. 1A) (23). As a first examination of how Mtb transcriptional/translational activity changes spatiotemporally as infection progresses, we thus infected C3HeB/FeJ mice with Mtb(P₆₀₆’::mKO-tetON, smyc’::mCherry) for 6, 12, or 16 weeks, followed by 1 week administration of doxycycline in the drinking water/food prior to sacrifice. Fixed lung samples were then analyzed for mKO signal in Mtb present in the lesion cuff versus core edge.

Consistent with our previous results (23), Mtb residing in the lesion core edge expressed high levels of mKO at 6 weeks post-infection (wpi) (Fig. 1, A to C), a timepoint when such lesions first form (23, 28), indicating an environment favorable for bacterial replication. Mtb residing in the cuff, in contrast, expressed substantially lower levels of mKO at 6 wpi (Fig. 1, A to C) (23). There was notably increased heterogeneity between lesions at 12 wpi, with some lesions exhibiting similar patterns of Mtb mKO expression as those at 6 wpi (Fig. 1D “lesion 1” and Fig. 1E) (i.e., higher levels of mKO in Mtb residing in the core edge versus those in the cuff), while other lesions had lower levels of mKO signal in Mtb residing in both the cuff and core edge (Fig. 1D, “lesion 5,” and Fig. 1E). These results suggested a downward transition in Mtb transcriptional/translational activity levels, particularly of bacteria in the lesion core edge, as infection progressed. Indeed by 16 wpi, the patterns of Mtb transcriptional/translational activity levels in the lesion cuff versus core edge was consistent across lesions, with Mtb expressing low levels of mKO in both the lesion core edge and cuff (Fig. 1, F and G). The variability in Mtb mKO signal within each lesion sublocation was also markedly decreased at 16 wpi as compared to 6 wpi, with the coefficient of variation (CV) for Mtb in the lesion cuff decreasing to 54.95% ± 2.69% from 117.32% ± 9.21%, P < 0.001, and to 45.27% ± 1.04% from 58.45% ± 3.16%, P < 0.01, for Mtb in the lesion core edge (compare Fig. 1C to G). These results indicate that Mtb transcriptional/translational activity decreases as lesions mature, with the lesion core edge becoming less favorable to bacterial replication as infection progresses.

Adaptation of hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) for detection of Mtb transcripts from in vivo samples

To understand how Mtb adapts to the changing environment as infection progresses and in spatial context, we pursued adaptation of hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) methodology to enable direct visualization of Mtb transcripts at the single bacterium level, in the context of intact tissue and lesion architecture. HCR-FISH is a quantitative approach where samples incubated with probes specific to an RNA of interest is proceeded by linear signal amplification via self-assembling fluorescently-labeled hairpins (Fig. 2A) (7, 11). As a first test of HCR-FISH on Mtb-infected lung samples, sectioned, paraffin-embedded lung tissue from C3HeB/FeJ mice infected for 13 weeks with Mtb constitutively expressing mCherry (smyc’::mCherry) were processed for analysis with probes against Mtb 16S ribosomal RNA (rRNA). Strong overlap between the 16S rRNA and mCherry signals was observed, supporting the specificity and utility of the 16S rRNA probes for marking all Mtb (Fig. 2B). However, the complex cell envelope of Mtb (27) presents a major hurdle for detection of messenger RNA (mRNA) transcripts, which are much less abundant than rRNA (35, 36), with standard permeabilization techniques ineffective in enabling sufficient probe penetration for Mtb mRNA visualization. To this end, we developed a stepwise procedure to systematically disrupt the different layers of the Mtb cell envelope (Fig. 2C, see Materials and Methods). First, deparaffinized lung sections were treated with amylase and pullulanase to digest Mtb capsule polysaccharides. This was followed by sequential treatment with sodium dodecyl sulfate (SDS) to disrupt envelope lipids, proteinase K and achromopeptidase to digest envelope proteins, and hydrochloric acid to disrupt the mycolic acid layer. Subsequently, treatment with recombinantly expressed and purified trehalose dimycolate (TDM) hydrolase from Mycobacterium smegmatis (37) and mycobacteriophage protein lysin B (38) target TDM and mycolylarabinogalactan, with a final lysozyme treatment for disruption of the peptidoglycan layer (Fig. 2C). Importantly, these treatments allowed for the detection of gene operons in Mtb (described below), while not affecting the ability to stain for host nuclei with DAPI, nor the robust detection of Mtb 16S rRNA. Indeed, these treatments resulted in not just brighter Mtb 16S rRNA signal, but also the increased detection of Mtb bacilli (fig. S1).

Fig. 2. — (A) Schematic depicting workflow of HCR-FISH for Mtb transcripts. Created in BioRender. Lawrence, A. (2026) https://BioRender.com/3eo8lua. (B) HCR-FISH probes against Mtb 16S rRNA effectively label all Mtb. 13 wpi mCherry Mtb-infected C3HeB/FeJ lung sample was probed with 16S rRNA (green) with HCR-FISH and imaged via confocal microscopy. (C) List of permeabilization treatments used to enable detection of Mtb mRNA, with the targets for each respective treatment noted. (D) Lesion overview at 6 wpi highlighting the 3 lesion sublocations analyzed for expression of different Mtb operons using HCR-FISH. Mtb 16S rRNA is shown in green and host nuclei stained with DAPI is shown in grayscale. (E) The lesion cuff is macrophage-dominant and the lesion core edge is neutrophil-dominant. 3D confocal images of the interface region of the lesion cuff and core edge from a 6 wpi mCherry Mtb-infected C3HeB/FeJ lung sample is shown. Ly6G (neutrophil) staining is shown in green, CD68 (macrophage) in blue, and DAPI staining of nuclei in grayscale. All Mtb are shown in red (*smyc’*::mCherry).

We focused our studies here on three timepoints from C3HeB/FeJ mice infections to capture spatiotemporal changes in Mtb transcriptional responses over the course of infection: (i) 2 weeks, which is prior to lesion formation, (ii) 6 weeks, when canonical necrotic lesions are first routinely detected (23, 28), and (iii) 16 weeks, which our studies with the inducible mKO reporter show substantial downregulation of Mtb transcriptional/translational activity compared to earlier timepoints. For spatial analysis, responses were categorized for Mtb residing in three distinct sublocations of canonical necrotic lesions: (i) the lesion cuff (predominantly intracellularly within macrophages), (ii) in the lesion core edge (a mixture of Mtb present within neutrophils and among necrotic cell debris), and (iii) in the lesion core center (extracellular Mtb) (Fig. 2, D and E) (23, 28). Responses of Mtb residing in cellular, non-necrotic, macrophage-rich lesions were additionally analyzed, to enable comparison of how the environment experienced by Mtb resident in macrophages present in the cuff of necrotic lesions may differ from those in non-necrotic lesions (23, 28). Notably, in a subset of lesions at 16 wpi, 16S rRNA signal was substantially weaker than the signal observed at earlier time points (fig. S2), suggesting that these bacteria may be in the process of being cleared by the host. For robustness of comparison, analysis of mRNA expression in the 16 wpi samples was restricted to those lesions that had similar levels of Mtb 16S rRNA expression as the earlier time points.

Spatiotemporal changes in expression of Mtb lipid utilization genes

Lipids, specifically cholesterol and fatty acids, are a critical nutrient source for Mtb and mutant Mtb strains that cannot utilize lipids are attenuated in vivo (3, 39, 40). To determine where and when Mtb expresses genes involved in lipid response, indicating exposure to and usage of lipids as a carbon source, we designed probes for rv3160c-rv3162c, part of the fundamental lipid response of Mtb (41, 42). At 2 wpi, Mtb resides primarily intracellularly within macrophages, and at this time point, low or non-detectable levels of rv3160c-rv3162c was observed in most bacteria (97.6% ± 1.2%), with very few, if any, bacteria expressing intermediate or high levels of this operon (Fig. 3, A and D, and fig. S3A). Lipid-rich foamy macrophages are not observed at this early timepoint (3), and this result directly supports that there is little exposure of Mtb to lipids as a primary carbon source at this early stage of infection.

Fig. 3. — (A to C) Representative 3D confocal images of Mtb at 2 wpi (A), 6 wpi (B), and 16 wpi (C) in C3HeB/FeJ mice, probed for the lipid response genes *rv3160c-rv3162c* (magenta). Mtb 16S rRNA labels all bacteria (green) and DAPI staining of host nuclei is shown in grayscale. Merged image is shown on the left, merged image without DAPI signal in the middle panel, and *rv3160c-rv3162c* signal is shown alone in the right panel (in grayscale for clarity). (D) *rv3160c-rv3162c* expression increases with time post-infection. The percentage of Mtb expressing different levels of *rv3160c-rv3162c* at 2 wpi and from the lesion cuff region at 6 and 16 wpi is shown, separated into 4 bins – non-detectable (ND), low (<250 signal/μm³), intermediate (250–500 signal/μm³), or high (>500 signal/μm³). (E and F) Mtb in the lesion core edge express lower levels of *rv3160c-rv3162c* at 6, but not 16, wpi. The percentage of Mtb in each lesion sublocation expressing different levels of *rv3160c-rv3162c* at 6 (E) and 16 (F) wpi is shown, separated into 4 bins—non-detectable (ND), low (<250 signal/μm³), intermediate (250–500 signal/μm³), or high (>500 signal/μm³). Data are from 5 mice for 2 wpi, from 5 lesions from 5 mice for 6 wpi, and from 5 lesions from 4 mice for 16 wpi, obtained across 3 independent experiments. P values were obtained with a 2-way ANOVA with Tukey’s multiple comparisons test in (D) to (F). Only significant comparisons are indicated. *P < 0.05, **P < 0.01, ****P < 0.0001.

At 6 wpi, rv3160c-rv3162c expression markedly increased in Mtb present in the lesion cuff (bacteria primarily intracellular in macrophages) (Fig. 3B, top row), with a significant increase in the percentage of Mtb expressing intermediate (250–500 signal intensity/μm³) and high (>500 signal intensity/μm³) levels of rv3160c-rv3162c (25.9% ± 2.8% combined, versus 2.4% ± 1.2% at 2 wpi, P < 0.0001) (Fig. 3, B, D, and E, and fig. S3B). A marked increase was similarly observed for Mtb present in the lesion core center (22.3% ± 4.2%) (Fig. 3, B and E). The necrotic core has previously been reported to be lipid-rich, due to high levels of necrotic host cells making up the caseum, including lipid-rich foamy macrophages (43). Interestingly, a greater percentage of Mtb present in the lesion core edge still had non-detectable levels of rv3160c-rv3162c transcript (23.2% ± 4.5% versus 6.9% ± 0.7% for the cuff, P < 0.0001; and versus 14.0% ± 2.2% for the core center, P < 0.05), with a correspondingly decreased percentage showing intermediate and high levels (Fig. 3, B and E, and fig. S3B). At 16 wpi, a high percentage of Mtb in all sublocations of necrotic lesions expressed rv3160c-rv3162c, with a trend of increased expression compared to the 6 wpi samples (Fig. 3, C, D, and F, and fig. S3C).

Finally, Mtb present in macrophage-rich non-necrotic lesions expressed lower levels of rv3160c-rv3162c compared to those residing in macrophages in the cuff of necrotic lesions, with the difference more marked at 16 wpi (9.3% ± 1.8% versus 33.2% ± 4.7% expressing intermediate or high levels combined, P < 0.01) (fig. S4). This result suggests that macrophage populations differ between those present in the cuff of necrotic lesions versus in non-necrotic lesions, with macrophages in non-necrotic lesions having decreased lipid accumulation and/or decreased lipid presence in a form available to Mtb, as compared to macrophages in the necrotic lesion cuff.

Together, these results reveal an overall transition of Mtb from a non-lipid-rich to lipid-rich local environment as necrotic lesions form. They further demonstrate how the dynamic nature of differences in Mtb localization alters its exposure to lipids, with spatial differences in the lesion core edge versus center during the early stages of lesion development that dissipate at later stages, as well as differences for Mtb resident in macrophages depending on localization in necrotic versus non-necrotic lesions.

Nitric oxide/hypoxia early responsive genes are upregulated by Mtb residing in the lesion cuff and core edge

As well as adaptation to changing nutrient sources, Mtb response to environmental cues vitally modulate bacterial growth. Nitric oxide (NO) and hypoxia represent two critical environmental cues able to drive Mtb into an adaptive non-replicating state, with upregulation of a set of genes known as the dormancy regulon (44–46). The two-component system DosRS(T) regulates the dormancy regulon, with the hspX operon most often used as a marker of the DosR-dependent hypoxia/NO-mediated transcriptional response (44–49). To gain insight into where and when Mtb encounters these critical stressors during infection, HCR-FISH probes were thus designed against the hspX operon genes. Expression of the hspX operon was detected even at 2 wpi, with only 9.5% ± 2.8% of Mtb with non-detectable probe signal (Fig. 4, A and D, and fig. S5A). This was initially surprising, as we had previously observed much greater expression of hspX at 4 versus 2 wpi in C57BL/6 J mice using a hspX’::GFP reporter Mtb strain (50). To follow up this finding, we performed HCR-FISH on the same infected tissue to test for expression of Nos2, which encodes inducible nitric oxide synthase (iNOS), the enzyme responsible for producing NO during Mtb infection (51). Consistent with Mtb hspX operon expression at 2 wpi, Nos2 was detected in the lungs of infected C3HeB/FeJ mice at this timepoint, with robust expression comparable to that observed in 6 wpi Mtb-infected lung samples (fig. S6).

Fig. 4. — (A to C) Representative 3D confocal images of Mtb at 2 wpi (A), 6 wpi (B), and 16 wpi (C) in C3HeB/FeJ mice, probed for the *hspX* operon (magenta). Mtb 16S rRNA labels all bacteria (green) and DAPI staining of host nuclei is shown in grayscale. Merged image is shown on the left, merged image without DAPI signal in the middle panel, and *hspX* operon signal is shown alone in the right panel (in grayscale for clarity). (D) The *hspX* operon is expressed throughout infection in Mtb residing in macrophages. The percentage of Mtb expressing different levels of the *hspX* operon at 2 wpi and from the lesion cuff region at 6 and 16 wpi is shown, separated into 4 bins—non-detectable (ND), low (<250 signal/μm³), intermediate (250–500 signal/μm³), or high (>500 signal/μm³). (E and F) Mtb in the lesion core edge express higher levels of the *hspX* operon at 6, but not 16, wpi, with expression very low in the lesion core center. The percentage of Mtb in each lesion sublocation expressing different levels of the *hspX* operon at 6 (E) and 16 (F) wpi is shown, separated into 4 bins—non-detectable (ND), low (<250 signal/μm³), intermediate (250–500 signal/μm³), or high (>500 signal/μm³). Data are from 5 mice for 2 wpi, from 5 lesions from 5 mice for 6 wpi, and from 5 lesions from 4 mice for 16 wpi, obtained across 3 independent experiments. P values were obtained with a 2-way ANOVA with Tukey’s multiple comparisons test in (D) to (F). Only significant comparisons are indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

At 6 and 16 wpi, robust hspX operon expression was also observed in a substantial percentage of Mtb residing in the lesion cuff, with 45.8% ± 3.4% and 57.7% ± 4.3% combined, respectively, expressing intermediate (250–500 signal intensity/μm³) or high (>500 signal intensity/μm³) levels (Fig. 4, B to D, and fig. S5, B and C). Intriguingly, the percentage of Mtb expressing the highest levels of the hspX operon was even greater in the Mtb population residing in the lesion core edge versus the lesion cuff at 6 wpi (30.3% ± 1.5% versus 18.9% ± 0.7%, P < 0.05) (Fig. 4E). As neutrophils produce NO and further consume oxygen in generation of the oxidative burst (18, 52, 53), it is possible that this finding reflects the presence of both NO and hypoxia in this lesion sublocation. This difference between the lesion cuff and core edge is however lost at 16 wpi, with the percentage of Mtb expressing the highest levels of the hspX operon in the lesion cuff increasing to now match that observed in the lesion core edge (28.0% ± 3.9% versus 27.1% ± 6.0%) (Fig. 4F).

Strikingly, we found that the hspX operon was expressed at notably lower levels in the necrotic core center, with only 4.7% ± 1.5% and 1.5% ± 0.3% of bacteria in this sublocation at 6 and 16 wpi, respectively, exhibiting the highest signal levels (Fig, 4, B, C, E, and F, and fig. S5, B and C). Importantly, although the hspX operon responds robustly to hypoxia, this initial strong response is not sustained, with the hspX operon not part of the characterized “enduring hypoxia response” (54). The high percentage (71.2% ± 2.1%) of Mtb residing in the necrotic lesion core with a low level (<250 signal intensity) of hspX operon expression at 16 wpi might thus reflect an extended exposure of that subpopulation of Mtb to hypoxia.

As with the lipid response, differences in hspX operon expression were also found between Mtb present in macrophages in non-necrotic lesions versus the cuff of necrotic lesions (fig. S7). In particular, only 6.8% ± 1.7% and 8.4% ± 3.8% of Mtb in the non-necrotic lesions exhibited the highest signal levels of the hspX operon at 6 and 16 wpi, respectively, compared to 18.9% ± 0.7% and 28.0% ± 3.9% of Mtb residing in the cuff of necrotic lesions (P < 0.001 and P < 0.05, 6 or 16 wpi, respectively) (fig. S7, A and C). Interestingly however, at 6 wpi, only 4.2% ± 1.9% of Mtb present in non-necrotic lesions had non-detectable levels of hspX operon expression, compared to 16.6% ± 2.9% of Mtb residing in the cuff of necrotic lesions (P < 0.05) (fig. S7A). These results indicate differences in timing, uniformity, and extent of Mtb exposure to NO and hypoxia between these sites, and reinforce differences that exist in the macrophage populations present in the cuff of necrotic lesions versus in non-necrotic lesions.

Together, these results show that Mtb is exposed to NO-related stress as early as 2 wpi in this infection model. The spatiotemporal differences in expression of the hspX operon both within necrotic lesions and between necrotic and non-necrotic lesions further reveals how Mtb residence in different immune cells or extracellularly alters its exposure to the vital environmental cues of NO and hypoxia.

ESX-1 Type VII secretion system effector genes are expressed most strongly by Mtb present in macrophages in the cuff of necrotic lesions and in non-necrotic lesions

Finally, in addition to undergoing shifts in metabolism and responding to environmental cues during infection, Mtb directly manipulates interactions with host immune cells via systems such as the ESX-1 type VII secretion system (55, 56). The ESX-1 type VII secretion system has been shown to be critical in Mtb infection (55–58), yet the timing and spatial regulation of these genes remains largely unknown. To examine expression of this critical virulence factor spatiotemporally, we thus designed probes against the espACD operon, which encode 3 important substrates of the ESX-1 secretion system (59–62). The majority of bacteria exhibited espACD expression even at 2 wpi, with just 9.7% ± 3.8% of Mtb with non-detectable levels (Fig. 5, A and D, and fig. S8A), supporting the importance of the ESX-1 type VII secretion system in host colonization by Mtb.

Expression of espACD in Mtb residing in the lesion cuff was similarly robust at 6 wpi, with just 9.8% ± 2.8% of Mtb with non-detectable levels (Fig. 5, B, D, and E, and fig. S8B). In contrast, at 6 wpi, espACD expression levels were significantly lower in Mtb residing in the lesion core edge, and particularly in the lesion core center, with a smaller percentage expressing intermediate or high levels of this operon versus Mtb residing in the lesion cuff (21.5% ± 3.3% and 17.9% ± 6.3% combined for the core edge and core center, respectively, versus 45.5% ± 6.7% for the cuff, P < 0.05 in each case) (Fig. 5, B, C, and E, and fig. S8B). Most bacteria in the lesion core edge and core center continued to express low or non-detectable levels of espACD at 16 wpi (Fig. 5, C and F, and fig. S8C). Interestingly, at 16 wpi, there was a downward trend of espACD expression in Mtb resident in the lesion cuff compared to 6 wpi, although this did not reach statistical significance (17.8% ± 2.4% with non-detectable levels versus 9.8% ± 2.8% at 6 wpi; 31.4% ± 3.9% with intermediate-high levels versus 45.5% ± 6.7% at 6 wpi) (Fig. 5D, and fig. S8, B and C).

Finally, in contrast to results with rv3160c-rv3162c and hspX operon expression, espACD expression was similar for Mtb present in macrophages in the cuff of necrotic lesions and in non-necrotic lesions, across both the 6 and 16 wpi timepoints (fig. S9).

Together, these findings provide key insight into the spatiotemporal expression of the ESX-1 type VII secretion system, indicating its importance particularly for Mtb resident in macrophages.

DISCUSSION

While the impact of heterogeneity during Mtb infection on disease outcome and treatment efficacy is now well-appreciated, with multiple studies employing single cell technologies focused on understanding host cell differences in spatiotemporal context (15–18), studies analyzing the bacterial aspect have posed a continued major technical challenge. The use of reporter strains and techniques such as laser capture microdissection combined with bulk RNA sequencing have been invaluable in providing first insight into Mtb physiology during infection (23, 48, 49, 63, 64). However, reporter strains that are encoded on episomal plasmids are limited to shorter-term infections and in their throughput, while laser capture microdissection-RNA sequencing approaches do not provide single cell resolution. More recently, FISH-based methods have been applied to Mtb but have been restricted to either highly expressed RNA (rRNA) or used many probes (120) together against several unrelated genes for Mtb mRNA detection (65–68). Our establishment here of HCR-FISH methodology for direct detection of single operons in individual Mtb in the context of intact lung tissue opens the path to spatiotemporally studying Mtb responses at the single bacterium level.

The spatial differences observed in our analysis of three key aspects of Mtb biology (lipid, NO/hypoxia, type VII secretion) in three distinct lesion sublocations (cuff, core edge, core center) and in macrophage-rich non-necrotic lesions (Fig. 6), enabled by the single bacterium resolution afforded by HCR-FISH, raise several intriguing questions for follow-up study. First, it highlights distinct differences in Mtb resident in macrophages (lesion cuff) versus in neutrophils (lesion core edge). This is particularly the case when lesions have first formed (6 wpi), with lower expression levels of the lipid response genes rv3160c-rv3162c and the ESX-1 type VII secretion system substrates espACD, and higher levels of the hspX operon, in Mtb residing in the lesion core edge versus cuff. While neutrophils can accumulate lipids (69, 70), how these levels compare to those in foamy macrophages and whether access of Mtb to lipids present in neutrophils versus in foamy macrophages differs is unknown. Mtb exhibits slowed growth when utilizing lipids as a carbon source (71, 72), and the lower levels of rv3160c-rv3162c expression of Mtb in the lesion core edge versus the cuff at 6 wpi is thus in accord with the higher Mtb transcriptional/translational activity in this sublocation revealed by the inducible mKO reporter experiment. While higher hspX operon expression appears counterintuitive to higher Mtb transcriptional/translational activity, it is important to note both that a similar phenomenon of active Mtb in neutrophils despite high hspX’::GFP reporter and inducible nitric oxide synthase expression has previously been observed in an early infection model of C57BL/6 J mice (73), and that the mKO range of Mtb in the lesion core edge at 6 wpi is very large, with clear bifurcation into transcriptionally/translationally active and inactive Mtb subpopulations in some cases (23). It will be interesting in future studies to multiplex readouts, to directly examine how hspX operon expression relates to Mtb activity and growth.

Considering the spatial differences in espACD expression (Fig. 6), while ESX-1-dependent neutrophil necrosis in vitro has been reported (74), the role of ESX-1 in Mtb-neutrophil interactions in vivo is largely unknown. The release of neutrophil extracellular traps (NETs) in a manner that does not result in neutrophil death and supports Mtb growth was recently discovered, and interestingly, strong staining of citrullinated histone 3 (NET marker) was observed in the region between the macrophage-dominant lesion cuff and the caseous center in Mtb-infected cynomolgus macaque lung samples (75). Notably, recent studies have begun to delineate the existence of different neutrophil subsets during Mtb infection that have differing impact on Mtb (69, 76–78). Mtb-neutrophil studies to date have largely focused on in vitro and early infection timepoints in animal models; as both Mtb growth status and utilization of lipids as a carbon source impact critically on treatment efficacy (23, 30–33, 79, 80), our findings here further draw attention to the need to understand how Mtb interaction with its host cell differs depending on host cell type/subtype, and between and within lesion sublocations, for effective therapeutic regimen design. Understanding how newly discovered neutrophil subsets may map onto the lesion context and the differences in Mtb physiology observed here will thus be important to examine in future studies.

Second, differences were also observed between Mtb residing in macrophages within the cuff of necrotic lesions versus those in non-necrotic lesions, with decreased exposure to lipids and NO/hypoxia for Mtb present in the latter sublocation (Fig. 6). Crosstalk between immune cell populations is becoming better appreciated during Mtb infection, with signaling between macrophages and neutrophils impacting their activation state and permissiveness to Mtb (75, 81, 82). As macrophages in necrotic lesions are directly proximal to the neutrophil-dominant lesion core edge, our results support the importance of the local environment for driving differences in immune cell subtype development. Combined with the diversity in immune cell composition observed among granulomas during human infection (17), mechanistic understanding of how immune cell cross-talk alters the local environment experienced by Mtb in spatial context is of particular interest for continuing studies.

Third, the striking low levels of hspX operon expression in Mtb present in the lesion core center compared to bacilli in the lesion cuff or core edge highlight the very different local environment experienced by extracellular Mtb in the lesion core center (Fig. 6). The core center of mature lesions has always been assumed to be hypoxic, even if direct determination has been technically precluded by the need for live host cells for positive pimonidazole labeling (18, 83, 84). While the hspX operon is widely used in the field as a marker of Mtb response to NO and hypoxia given its extremely strong upregulation upon initial exposure (on the order of hours) to these signals (44–46), it is crucial to note that this upregulation decreases with time. Indeed, a different set of genes has been described to mark an extended response to hypoxia (54), and further studies examining members of this “enduring hypoxia response” gene set will be needed to robustly define how oxygen availability within the lesion core center changes as infection progresses.

Temporally for bacteria residing in macrophages, our results provide direct bacterial response data supporting the switch to lipid metabolism as infection progresses, in line with foamy macrophage formation as necrotic lesions form. At the same time, the increase in hspX operon and downward trend in espACD signals for Mtb residing in the lesion cuff from 6 to 16 wpi raise questions as to whether there is a transition of the macrophage subtype in which the bacteria reside as infection progresses. Interstitial macrophages are known to be more restrictive for Mtb growth versus alveolar macrophages (73), but how they may differentially contribute to lesion composition in spatiotemporal context, as well as the provenance of the abundant foamy macrophages found in the necrotic lesion cuff, is not well-understood. The significant decrease in variation in Mtb transcriptional/translational activity status in the lesion cuff with time (decrease in coefficient of variation of the inducible mKO signal) further points to changes in Mtb-host cell interplay as infection progresses and lesions mature. Together, these findings reinforce the need to understand spatiotemporal changes in bacterial responses in treatment design, particularly as therapy must successfully target established, not just beginning, infections, for effectiveness against a chronic disease like tuberculosis.

The HCR-FISH methodology for Mtb mRNA detection presented here enables a straightforward and accessible approach for testing the expression of specific Mtb operons of interest in spatial tissue/lesion context. We propose that future studies building on the foundational framework established here will continue to provide crucial insight into what drives Mtb population heterogeneity during infection and facets vital to bacterial survival and adaptation to specific local niches. For example, multiplexing different Mtb operons that reflect the bacterial response to different signals, with host aspects, or with use of the FISH-based “RS ratio” [ratio of short-lived spacer rRNA region to stable mature rRNA, which serves as a proxy for rRNA synthesis activity that tightly correlates with bacterial replication (65, 67, 85)], will allow direct in situ analysis of the relationships between Mtb environmental response, growth, and host cell/location phenotype. The C3HeB/FeJ murine infection model used in our studies here is well-appreciated for its utility for Mtb-host interaction studies, given the recapitulation of key lesion types observed during human infection (28, 29). Nonetheless, as the bacterial load in a typical necrotic lesion in C3HeB/FeJ mice is higher than that found in other models such as non-human primates, follow-up studies with HCR-FISH methodology in these other less permissive models will be of interest as well. Utilization of HCR-FISH methodology in combination with drug treatment or specific perturbation of Mtb regulatory pathways further holds immense promise in revealing how drug efficacy is affected spatiotemporally, and how targeting of key nodes in Mtb signal integration can be leveraged for altering infection heterogeneity to aid treatment success.

Finally, the HCR-FISH approach established here can also be used to directly probe tissue samples from patients, potentially opening a path to the study of not just the most common pulmonary manifestation of Mtb infection, but also disease outcomes with poor animal models. We additionally anticipate that this approach can be broadly adapted to other difficult to permeabilize bacteria for which FISH-based detection of mRNA have also posed technically challenging, such as Streptococcus pneumoniae and Clostridiodes difficile, to excitingly enable interrogation of the impact of spatiotemporal heterogeneity across multiple other important infectious diseases.

MATERIALS AND METHODS

Ethics statement

Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts University (#B2024–90), in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care, the US Department of Agriculture, and the US Public Health Service guidelines. These protocols followed standards set by the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.”

Mtb strains and culture

Mtb Erdman wild type was used in this study. Edman(P₆₀₆’::mKO-tetON, smyc’::mCherry) has been previously described (23). Preparation of mouse infection stocks was as previously described (48).

Mouse Mtb infections

6 week old female C3HeB/FeJ wild type mice (Jackson Laboratory #000658, Bar Harbor, ME) were intranasally infected with 10³ colony forming units of Mtb in 35 μl of phosphate-buffered saline (PBS) containing 0.05% Tween-80, under light anesthesia with 2% isoflurane (23, 48, 49). Mice were sacrificed at 2, 6, 7, 13, 16, or 17 wpi and lungs fixed in 4% paraformaldehyde (PFA) in PBS overnight at room temperature, then stored in phosphate buffered saline (PBS) supplemented with 100 U SUPERase·In RNase inhibitor (ThermoFisher Scientific, Waltham, MA) prior to further processing. For the P₆₀₆’::mKO-tetON, smyc’::mCherry infections, one week prior to sacrifice, either drinking water containing 1 mg/ml doxycycline with 5% sucrose or food containing 200 mg/kg doxycycline (Bioserv, Flemington, NJ) were supplied to the mice (23, 86).

Recombinant protein expression and purification

Plasmids encoding TDM hydrolase or lysin B (37, 38) were expressed in E. coli BL21(DE3) in the presence of 100 μg/ml ampicillin. For TDM hydrolase, 1 L LB broth was inoculated directly with a colony from a fresh transformation plate and grown to OD₆₀₀ ∼ 0.6 prior to induction with 1 mM IPTG for 3 hours at 30°C. For lysin B, overnight cultures were diluted into 1 L LB broth and grown to OD₆₀₀ ∼ 0.6 prior to induction with 1 mM IPTG for 4 hours at 37°C. Cells were pelleted and resuspended in low imidazole buffer (500 mM NaCl, 50 mM Tris pH 7.5, 15 mM imidazole, 10% glycerol) before being flash-frozen in liquid nitrogen and stored at −80°C prior to purification. Thawed cells were lysed by sonication, in the presence of protease inhibitors (Pierce mini protease inhibitor tablets, ThermoFisher Scientific, Waltham, MA). The soluble fraction was collected and incubated overnight with 1 ml nickel-NTA agarose beads (Machery-Nagel, Dueren, Germany) at 4°C, with agitation. To elute the protein, beads were washed 3x with low imidazole buffer prior to elution in high imidazole buffer (500 mM NaCl, 50 mM Tris pH 7.5, 200 mM imidazole, 10% glycerol). Samples were dialyzed overnight at 4°C to remove imidazole using Slide-A-Lyzer G3 dialysis cassettes (ThermoFisher Scientific, Waltham, MA) in 50 mM Tris pH 7.5, 100 mM NaCl, 25 mM MgCl₂ (TDM hydrolase), or in 50 mM Tris pH 8, 50 mM NaCl (lysin B) (37, 38). Protein was collected from the dialysis cassettes the next day, aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C until use.

HCR-FISH on Mtb transcripts

For HCR-FISH analyses, lung samples were processed for paraffin embedding and sectioning (5 μm thick sections) (Tufts Comparative Pathology Services) and stored at 4°C in air-tight containers with desiccant until use. Paraffin was removed by washing with xylene substitute (Fisher Scientific, Waltham, MA) for 3 x 5 minutes, followed by rehydration using decreasing concentrations of ethanol from 100% to 50% for 3 minutes each, with a final wash step in UltraPure water (ThermoFisher Scientific, Waltham, MA) for 3 minutes. Samples were then permeabilized to detect Mtb transcripts, via sequential treatment in a humidified chamber with: (i) 1 μg/ml amylase (MilliporeSigma #A6380, Burlington, MA) and 1:1000x dilution of pullulanase (MilliporeSigma #E2412, Burlington, MA) in 20 mM sodium acetate, pH 5 buffer for 1 hour at 37°C; (ii) 1% SDS in PBS for 15 minutes at room temperature; (iii) 10 μg/ml proteinase K (Fisher Scientific #BP1700–100, Waltham, MA) in 10 mM Tris HCl pH 7 for 10 minutes at 37°C; (iv) 30 U achromopeptidase (MilliporeSigma #A3422, Burlington, MA) in 10 mM Tris HCl pH 8.5 for 40 minutes at 37°C; (v) 0.2 M HCl for 2.5 minutes at 37°C; (vi) TDM hydrolase and lysin B at 1 mg/ml and 1.5 mg/ml respectively in 50 mM Tris pH 8, 100 mM NaCl and 25 mM MgCl₂ for 2 hours at 37°C; and (vii) 10 mg/ml lysozyme (Roche #10837059001, Basel, Switzerland) in TE buffer pH 7 for 1 hour at 37°C. All treatments were carried out in 100 μl volumes, and 200 U SUPERase·In RNase inhibitor (ThermoFisher Scientific, Waltham, MA) was added to inhibit RNase activity during permeabilization. Following permeabilization, antigen retrieval was performed in Tris-EDTA buffer at 95°C for 15 minutes, after which slides were cooled to 45°C in 20 minutes by adding 40 ml UltraPure water every 5 minutes. Slides were further incubated in 50 ml UltraPure water at room temperature for 10 minutes prior to 2 x 2 minute washes with PBS containing 0.1% Tween-20 (PBST).

For probe hybridization, the prepared tissue slices were first incubated with 200 μl probe hybridization buffer (Molecular Instruments, Los Angeles, CA) with 0.1 mg/ml salmon sperm DNA (ThermoFisher Scientific, Waltham, MA) for 10 minutes prior to addition of probes at 0.02 μM concentration overnight in probe hybridization buffer at 37°C in a humidified chamber. Probes were obtained from Molecular Instruments (Los Angeles, CA), and were designed against Mtb 16S rRNA (labels all bacteria), rv3160c-rv3162c (lipid-responsive), the hspX operon (rv2028c-rv2031c) (hypoxia/nitric oxide-responsive), and the espACD (rv3614c-rv3616c) operon (type VII secretion system substrates), using sequences from the Mtb Erdman strain genome (NCBI Genbank accession AP012340.1). The following day, unbound probes were removed by washing with decreasing concentrations of probe wash buffer (Molecular Instruments, Los Angeles, CA) (100% to 0%) diluted in 5x saline-sodium citrate buffer containing 0.1% tween-20 (SSCT) at 37°C for 15 minutes each, followed by one wash at room temperature with 5x SSCT for 5 minutes. Tissue sections were then treated with 200 μl amplification buffer containing 0.1 mg/ml salmon sperm DNA for 30 minutes at room temperature. At the same time, 4 μl of 3 μM hairpin stock per slide were heated at 95°C for 90 seconds in a thermocycler and then cooled down to RT, protected from light, for 30 minutes. To detect RNA in tissue, Alexa Fluor 488 and 647 amplifiers were used for 16S rRNA and mRNA target detection, respectively. Amplifier fluorophores in amplification buffer were added directly to the tissue samples prior to overnight incubation at room temperature. The next day, slides were first washed with 5x SSCT for 5 minutes, then again with 5x SSCT for 2 x 15 minutes, with a final 5x SSCT wash for 5 minutes. Samples were stained with DAPI (ThermoFisher Scientific, Waltham, MA) diluted 1:500 in blocking buffer (3% BSA + 0.1% triton X-100 in PBS) for 10 minutes at room temperature. Slides were washed 3x with 5x SSCT prior to mounting with Prolong Glass (ThermoFisher Scientific, Waltham, MA). Coverslips were allowed to cure overnight at room temperature prior to imaging.

For initial comparative tests of permeabilization efficacy, HCR-FISH was initially performed without the added permeabilization steps, with antigen retrieval and probe hybridization proceeding immediately after tissue rehydration. After the final wash steps, slides were mounted with VectaShield mounting media (Vector Laboratories, Newark, CA) and imaged using a Leica SP8 spectral confocal microscope. Coverslips were then removed by immersing the slides in 50 ml PBS for 15 minutes. The same samples then underwent all permeabilization steps as described above. Following the permeabilization treatments, slides were incubated with probe hybridization buffer and relabeled with 16S rRNA probes. After signal amplification, tissue sections were washed and mounted with Prolong Glass (ThermoFisher Scientific, Waltham, MA). The same regions were imaged with identical laser settings to compare signal intensity before and after permeabilization.

HCR-FISH on host transcripts

To detect expression of Nos2 in Mtb-infected lungs, paraffin-embedded sections were processed and stained as described above, but excluding the additional permeabilization steps required for Mtb permeabilization. Briefly, after tissue rehydration, lung sections underwent antigen retrieval at 95°C for 15 minutes prior to allowing the samples to cool. Slides were treated with 2 x 2 minute PBST washes followed by probe hybridization. Signal amplification was performed as described for Mtb transcripts.

Confocal microscopy and image quantification

For the P₆₀₆’::mKO-tetON, smyc’::mCherry reporter infections, lung lobes were embedded in 4% agarose in PBS and 250 μm thick sections obtained using a Leica VT1000S vibratome (23, 87). Lung sections were blocked with 3% BSA + 0.1% Triton X-100 in PBS (blocking buffer) for 1 hour at room temperature (all steps protected from light) before overnight incubation with rat anti-CD68 (Bio-Rad #MCA1957, Hercules, CA) primary antibody at 1:100 dilution in blocking buffer at room temperature. The following day, lung samples were washed 3 x 5 min with blocking buffer prior to staining with Alexa Fluor 647 goat anti-rat secondary antibody (ThermoScientific, Waltham, MA; 1:100 dilution) and DAPI (ThermoScientific, Waltham, MA; 1:500 dilution) for 2 hours at room temperature in blocking buffer. Tissue slices were mounted using VectaShield mounting medium (Vector labs, Newark, CA). Where macrophages and neutrophils were visualized together, rabbit anti-CD68 (Cell Signaling Technology #97778, Danvers, MA; 1:200 dilution) and rat anti-Ly6G (BD Pharmingen #551459, Woburn, MA; 1:100) primary antibodies were used. Samples were imaged on a Leica SP8 spectral confocal microscope using a 40x oil immersion objective. z-stacks were 10 μm thick with 0.5 μm z-steps. Images were reconstructed into 3D using Volocity software (Quorum Technologies, Inc., Ontario, Canada). HCR-FISH samples were imaged using a Leica SP8 spectral confocal microscope with z-stacks that were 3 μm thick with 0.35 μm z-steps. For all microscopy analyses, regions were selected based on the presence of bacteria without regard to reporter or mRNA signal, and lesions across multiple mice and multiple experiments sampled. Lesion sublocations were defined by the host cell DAPI staining status (see Fig. 1A, Fig. 2, D and E). Specifically, the “core edge” is marked by dense DAPI staining with the distinct presence of many multi-lobed nuclei representative of neutrophils. External to this core edge is the lesion “cuff”, marked by intact host nuclei that is less densely packed. The “core center” is marked by a lack of intact nuclei, and is internal to the core edge.

Quantification of mKO reporter and mRNA signal was performed as previously described using Volocity software (23, 48, 49, 87). Briefly, Mtb bacilli were identified by either mCherry signal (for the mKO reporter infection) or 16S rRNA signal (for HCR-FISH studies) using signal intensity and size thresholding (excluding “objects” ≤1 μm³, which removes background objects that are not actual Mtb cells). Each “object” was individually verified to be a single bacterium and in cases where an identified “object” was comprised of multiple bacteria, manual cropping was performed to separate the bacterial cells. In heavily-infected host cells where manual cropping into individual bacteria was not feasible, the “separate touching objects” function in Volocity was utilized, with any bacterial cell that possessed distinct mKO or mRNA signal within the tight cluster cropped out first to ensure accurate analysis. The total signal (either mKO or mRNA probes of interest) within each bacterium was then measured and analyzed as signal/μm³ (volume of the bacterium) to normalize for differences in bacterial size. Settings for mKO and each mRNA target were maintained across samples to allow for direct comparison across lesions and timepoints. For HCR-FISH analysis, normalization for differences in background signal was also performed, with the background fluorescence intensity/μm³ of 8 random locations not containing bacteria measured and subtracted from the HCR-FISH signal for each image.

Statictical analysis

Details of data quantification and statistical analysis utilized are described in the figure legends. Statistical tests were computed using GraphPad Prism software (Boston, MA). A 2-way ANOVA with Tukey’s multiple comparisons test was utilized in Fig. 3, D to F, Fig. 4, D to F, and Fig. 5, D to F. Unpaired t-tests with Holm-Sidak multiple comparisons test was utilized in fig. S4, A and C, fig. S7, A and C, and fig. S9, A and C. P < 0.05 was considered significant in all cases.

Acknowledgments

We thank G. Hatfull and A. Ojha for the kind gift of plasmids for recombinant expression and purification of lysin B and TDM hydrolase, respectively. We thank M. Sandkvist and D. Lawrence for technical advice regarding recombinant expression of TDM hydrolase, and members of the Tan lab for helpful discussion.

Funding:

National Institutes of Health grant R01 AI143768 (S.T.)., Hypothesis Fund (S.T.)., National Institutes of Health grant F32 AI188687 (A.E.L.).

Author contributions:

Conceptualization: A.E.L., S.T., Methodology: A.E.L., S.T., Formal analysis: A.E.L., S.T., Investigation: A.E.L., S.T., Writing—original draft: A.E.L., S.T., Writing— review and editing: A.E.L., S.T., Supervision: S.T., Funding acquisition: S.T.

Competing interests:

The authors declare that they have no competing interests.

Data, code, and materials availability:

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. The plasmid for recombinant expression and purification of lysin B or TDM hydrolase can be provided by S.T. pending scientific review and a completed material transfer agreement with Dr. Graham Hatfull (University of Pittsburgh) or Dr. Anil Ojha (Wadsworth Center), respectively. Requests for these plasmids should be submitted to: S.T. at shumin.tan@tufts.edu.

Supplementary Materials

This PDF file includes:

Figs. S1 to S9

sciadv.aeb8377_sm.pdf^{(6.3MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S9

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Data Availability Statement

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PERMALINK

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

Anna-Lisa E Lawrence

Shumin Tan

Roles

Abstract

INTRODUCTION

RESULTS

Mtb transcriptional/translational activity within necrotic lesions decreases as infection progresses

Fig. 1. Mtb transcriptional/translational activity in the necrotic lesion core edge decreases as infection progresses.

Adaptation of hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) for detection of Mtb transcripts from in vivo samples

Fig. 2. Adaptation of HCR-FISH for detection of Mtb transcripts in the context of intact lung tissue.

Spatiotemporal changes in expression of Mtb lipid utilization genes

Fig. 3. Spatiotemporal changes in expression of Mtb lipid utilization genes.

Nitric oxide/hypoxia early responsive genes are upregulated by Mtb residing in the lesion cuff and core edge

Fig. 4. Nitric oxide/hypoxia early responsive genes are upregulated by Mtb residing in the lesion cuff and core edge.

ESX-1 Type VII secretion system effector genes are expressed most strongly by Mtb present in macrophages in the cuff of necrotic lesions and in non-necrotic lesions

Fig. 5. ESX-1 type VII secretion system effector genes are expressed most strongly by Mtb present in the lesion cuff and decreases with time.

DISCUSSION

Fig. 6. Summary of spatiotemporal environmental adaptation of Mtb in the lung.

MATERIALS AND METHODS

Ethics statement

Mtb strains and culture

Mouse Mtb infections

Recombinant protein expression and purification

HCR-FISH on Mtb transcripts

HCR-FISH on host transcripts

Confocal microscopy and image quantification

Statictical analysis

Acknowledgments

Funding:

Author contributions:

Competing interests:

Data, code, and materials availability:

Supplementary Materials

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REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

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