Trans-species communication in the Mycobacterium tuberculosis-infected macrophage

Abstract

Much of the infection cycle of Mycobacterium tuberculosis (Mtb) is spent within its host cell, the macrophage. As a consequence of the chronic, enduring nature of the infection, this cell-cell interaction has become highly intimate, and the bacterium has evolved to detect, react to, and manipulate the evolving, immune-modulated phenotype of its host. In this review, we discuss the nature of the endosomal/lysosomal continuum, the characterization of the bacterium's transcriptional responses during the infection cycle, and the dominant environmental cues that shape this response. We also discuss how the metabolism of both cells is modulated by the infection and the impact that this has on the progression of the granuloma. Finally, we detail how these transcriptional responses can be exploited to construct reporter bacterial strains to probe the temporal and spatial environmental shifts experienced by Mtb during the course of experimental infections. These reporter strains provide new insights into the fitness of Mtb under immune- and drug-mediated pressure.

Keywords: Mycobacterium tuberculosis; macrophage; phagosome; tuberculosis.

Fig. 1. Real-time measurement of changing phagosomal hydrolase activity in the phagosome during the maturation process

Assays have been developed that measure a range of lysosomal hydrolase activities including bulk proteinase activity, cysteine proteinase activity, lipolysis and β-galactosidase activity. The assays involve fluorogenic substrates that are linked to silica beads (A). These beads are also coupled with an opsonizing molecule, such as IgG or mannosylated BSA, to facilitate their uptake by macrophages, and a calibration fluorophore. Results are usually expressed as a ratio of substrate fluorescence:calibration fluorescence. The examples illustrated show experiments with beads coupled with the proteinase substrate DQ Green BSA, which consists of albumin derivatized with a self-quenching fluorophore. The ensuing change in fluorescence generated through the release of fluorescent peptides can be measured by several different platforms, each of which provides its own unique insight. A spectrofluorometer (B) provides a kinetic readout that represents an average value across a population of cells mounted on glass coverslips in cuvettes. A confocal microscope (C) allows visualization and quantification of the increase in green fluorescence from the hydrolyzed substrate compared with the red fluorescence of the calibration fluorophore at the level of the individual phagosome. These frames are from a 45-minute movie. Flow cytometry (C) enables examination of the heterogeneity of the activity across the cell population at the level of each individual cell. Analysis by flow cytometry can be combined with immunofluorescence with antibodies against pathogens, or cell surface markers. This figure was modified from Russell et al. (5).

Fig. 2. Life and death dynamics during long-term intracellular survival of Mtb.

(A) Bacterial survival assays. Resting murine bone-marrow-derived macrophages were infected at low multiplicity of infection (~1:1) with CDC1551. Viable CFUs were quantified at day 0 and at 2-day intervals post-infection over a 14-day time course by lysis of monolayers, serial dilution, and plating on 7H10 medium. Error bars indicate standard error of the mean from two independent biological replicates, each consisting of three technical replicates per strain (total of six wells/strain). (B) Replication clock plasmid. The percentage of bacteria containing the pBP10 plasmid during growth in resting macrophages was determined by comparing CFUs (mean ± SD) recovered on kanamycin versus non-selective media (red). The cumulative bacterial burden (CBB) (black) was determined by mathematical modeling based on total viable CFUs and plasmid frequency data. Data shown represent two independent experiments, with each sample performed in quadruplicate (eight total wells/time point). (C) The ‘bottleneck’ response. Temporal expression profiles of genes differentially regulated at day 2 post-infection, shown as ratio of signal intensity relative to control. Note the maximal change in transcript levels at day 2 post-infection followed by the majority trending back toward control levels. (D) ‘Guilt by association’ analysis. Genes regulated in synch with known virulence regulons (i.e. the DosR regulon) were identified by using a highly regulated member of this regulon, hspX. This figure is reproduced from Rohde et al. (43)

Fig. 3. Mtb utilizes pH and [Cl⁻] as environmental cues

(A) Phagosome acidification is a key inducer of Mtb transcriptional changes. Murine bone marrow-derived macrophages were infected with CDC1551 for 2 h. For the test set, the macrophages were treated with 100 nM concanamycin A (CcA) to inhibit phagosomal acidification, before Mtb infection. Y-axis shows gene expression ratios from Mtb present in untreated phagosomes (pH 6.4) relative to CcA-treated phagosomes (pH 7.0). Mtb genes whose induction at 2 h was sensitive to CcA (>1.5-fold, p < 0.05) are shown in red. Red arrows indicate examples of genes induced at 2 h in untreated macrophages whose expression was insensitive to CcA. (B) [Cl⁻] and pH are inversely correlated during phagosome maturation. 10,10′-Bis[3-carboxylpropyl]-9,9′-biacridinium (BAC)/pHrodo beads were added to murine bone marrow-derived macrophages and BAC (green) and pHrodo (red) fluorescence tracked with a microplate reader over time. F₀ is fluorescence at time = 0 min, and F is fluorescence at each given time point. BAC fluorescence decreases as [Cl⁻] increases. pHrodo signal increases as pH decreases. Data are shown as means ± SD from 4 wells. (C) Links between Mtb’s response to pH and Cl⁻. The indicated Mtb strains were grown in broth buffered at pH 7.0, pH 7.0 + 250 mM NaCl, pH 5.7, or pH 5.7 + 250 mM NaCl for 4 h. qRT-PCR of rv2390c expression in WT, ΔphoPR, and the complemented mutant (phoPR*) is shown. Fold induction is as compared to the corresponding strain grown in media at pH 7.0. Data are shown as means ± SD from 3 technical replicates. This figure is reproduced from Rohde et al. (23) and Tan et al. (54).

Fig. 4. Expression of the lipid droplet-associated protein Peripilin 2 in human tuberculosis granulomas

Immunofluorescence signals were obtained for each granuloma (B), and the corresponding region from a hematoxylin and eosin stained slide (A) is shown. Nuclei are shown in blue and antigens in red. The macrophages subtending the caseum of this fibrocaseous granuloma label strongly for peripilin 2 expression. This figure is reproduced from Kim et al. (94).

Fig. 5. Impact of host immune response on dynamics of hspX′::GFP induction

Erdman(hspX′::GFP, smyc′::mCherry) was inoculated into vaccinated or mock-treated C57BL/6J WT mice for up to 28 days. 3D confocal images from a 14-day infection are shown. All bacteria are marked in red (smyc′::mCherry), reporter signal is shown in green (hspX′::GFP), nuclei are marked in grayscale (DAPI), and phalloidin staining of f-actin is shown in blue. Scale bar 10 μm. Graph shows quantification of the GFP/μm³ signal for each bacterium measured from multiple 3D confocal images, at the indicated time points. Each point on the graph represents a bacterium or a tightly clustered group of bacteria (mock-treated – filled symbols, vaccinated – open symbols; WT mice infections – black, NOS2^−/− mice infections - blue). Horizontal lines mark the median value for each group. p-values were obtained with a Mann-Whitney statistical test. This figure is reproduced from Sukumar et al. (112).

Fig. 6. SSB-GFP reporter tracks Mtb replication in vivo

Erdman(SSB-GFP, smyc′::mCherry) was inoculated into vaccinated or mock-treated C57BL/6J WT mice for up to 28 days. 3D confocal images from a 28-day infection are shown with all bacteria marked in red (smyc′::mCherry), reporter signal shown in green (SSB-GFP), nuclei marked in grayscale (DAPI), and phalloidin staining of f-actin shown in blue. For clarity of foci visualization, SSB-GFP signal is shown in extended focus, overlaid on the 3D image. Scale bar 10 μm. Graph shows the percentage of Mtb displaying SSB-GFP foci for each mouse, measured from multiple 3D confocal images, at 14 and 28 days post-challenge. Each point on the graph represents a mouse (mock-treated – filled symbols, vaccinated – open symbols). Horizontal lines mark the median value for each group. p-values were obtained with a Mann-Whitney statistical test. This figure is reproduced from Sukumar et al. (112).