A novel mycobacterial <i>In Vitro</i> infection assay identifies differences of induced macrophage apoptosis between CD4⁺ and CD8⁺ T cells

<div><p>Macrophages are natural host cells for pathogenic mycobacteria, like <i>Mycobacterium tuberculosis (M</i>.<i>tb)</i>. Immune surveillance by T cells and interaction with <i>M</i>.<i>tb</i> infected macrophages is crucial for protection against <i>M</i>.<i>tb</i> reactivation and development of active tuberculosis. Several factors play a role in the control of <i>M</i>.<i>tb</i> infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional <i>in vitro</i> assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional <i>in vitro</i> assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a <i>Mycobacterium bovis</i> BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated <i>in vitro</i> and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. <i>M</i>.<i>tb</i> protein specific CD4⁺ and CD8⁺ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4⁺ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional <i>in vitro</i> assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.</p></div

<i>In vitro</i> generation of effector T cells (E) and co-culture with LD-BCG infected MDM (M) at different E/M ratios.

<p>(A) Workflow depiction for the generation of effector T cells, LD-BCG infected MDM, E/M co-culture, flow cytometry analyses of infected MDM and effector T cells. (B) Analyses of MDM infected with LD-BCG after co-culture with effector T cells stimulated with <i>Staphylococcus</i> Enterotoxin B (SEB), <i>M</i>. <i>tuberculosis</i> Purified Protein Derivative (PPD), and without stimulation (w/o). Different E/M ratios are shown on the x-axes. Proportions of infected MDM with live (grey) or dead LD-BCG (open) are shown as stacked boxes (left graph). Absolute numbers of infected MDM are shown as symbols (middle graph) and numbers of MDM infected with live LD-BCG are shown as boxes (right graph). The dotted line in the middle graph indicates MDM numbers infected with LD-BCG w/o effector T-cell co-culture. Median with range of triplicates from a representative experiment are depicted.</p

Proportional and absolute differences of LD-BCG infected MDM and comparison with Colony Forming Units (CFU) and fluorescence microscopy.

<p>Rifampicin treatment of MDM infected with LD-BCG at different indicated concentrations is shown. (A) Flow cytometry (FACS) analysis of MDM proportions infected with live (grey) or dead (open) LD-BCG are shown as stacked boxes. (B) Comparison of absolute MDM numbers infected with LD-BCG measured by bead corrected flow cytometry. Bead-based standardization of measured sample volume is shown in the upper graphs. Absolute numbers of all infected MDM (lower left graph) as well as MDM containing live and dead LD-BCG (lower right graph) at different rifampicin concentrations are shown. Median with range of triplicates are depicted. (C) Comparison of live LD-BCG infected MDM numbers measured by FACS and mycobacterial culture (CFU). Circles indicate FACS values adjusted for multiple infections as determined by fluorescence microscopy. Median values with range of triplicates are depicted. (D) Fluorescence microscopy analyses of MDMs infected with LD-BCG with or w/o ATC or non-infected MDM are shown. Blue color indicates MDM nuclei; red color indicates mCherry expressing LD-BCG; green color indicates GFP-expressing live LD-BCG. A representative experiment of three is shown.</p

Cytotoxic effects of total effector T cells and T-cell subpopulations against LD-BCG infected MDM and markers of induced cell death on MDM.

<p>Combined analyses of autologues E/M samples from healthy donors (n = 8) for (A), (n = 6) for (B), and (n = 5) for (C) are shown. Total effector T cells (A) or enriched CD4⁺/CD8⁺ T-cell subpopulations (B, C) were applied. (A, B) Cytotoxicity of differentially stimulated effector T cells against MDM infected with LD-BCG was assessed by flow cytometry. Different E/M ratios were applied (x-axes) and absolute numbers of MDM infected with live LD-BCG are indicated (y-axes). Mean and standard deviations are shown. (C) Apoptosis marker expression on MDM with or w/o LD-BCG infection after coculture with differentially stimulated CD4⁺ or CD8⁺ effector T cells. E:M ratios of 1:3 are shown. The proportions of non-apoptotic (open), early apoptotic (bright grey), and late apoptotic (dark grey) MDM are depicted as pie charts. Non-infected MDM (left panel) as well as MDM infected with live (middle) and dead (left) LD-BCG are shown. Median values of five independent experiments are depicted. The Mann-Whitney U-test were applied. Asterisks indicate significant differences (***: p<0.001; **: p<0.01; *: p<0.05).</p

Comparison of expression of apoptotic markers induced by differentially activated CD4⁺ and CD8⁺ T cells in MDM with live BCG (E:M 1:1)

<p>Comparison of expression of apoptotic markers induced by differentially activated CD4⁺ and CD8⁺ T cells in MDM with live BCG (E:M 1:1)</p

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of <i>Mycobacterium tuberculosis</i>

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus <i>Mucor irregularis</i>, which was obtained from the Cameroonian medicinal plant <i>Moringa stenopetala</i>. Chlorflavonin exhibited strong growth inhibitory activity <i>in vitro</i> against <i>Mycobacterium tuberculosis</i> (MIC₉₀ 1.56 μM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 μM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against <i>Mycobacterium tuberculosis</i> inside infected macrophages was demonstrated and was superior to streptomycin treatment

Probing the Mycobacterial Trehalome with Bioorthogonal Chemistry

Mycobacteria, including the pathogen <i>Mycobacterium tuberculosis</i>, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome

Revised model of GlgE-mediated intracellular and capsular α-glucan synthesis in mycobacteria.

<p>The M1P building block for the maltosyltransferase GlgE is formed <i>via</i> two alternative routes, TreS-Pep2 and GlgC-GlgA. Both routes are connected <i>via</i> the shared use of ADP-glucose by GlgA and OtsA, the latter providing the trehalose substrate for the TreS-Pep2 pathway. GlgA, like OtsA, is also capable of using UDP-glucose as a donor, which in turn is produced from G1P by GalU, but this appears to be less significant <i>in vivo</i>. GlgE is essential for intracellular and capsular α-glucan synthesis and generates linear maltooligosaccharides as the substrates for the branching enzyme GlgB. α-Glucans are produced intracellularly with partial coupling to secretion by unknown transport mechanisms. Steps highlighted in red are new findings as described in this report. G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; M1P, α-maltose 1-phosphate; T6P, trehalose 6-phosphate; ADPG, ADP-glucose; UDPG, UDP-glucose.</p

The central importance of the GlgE pathway in intracellular and capsular α-glucan synthesis in <i>M</i>. <i>tuberculosis</i>.

<p>Quantification of intracellular (A) or extracellular (i.e. capsular) (B) α-glucan in <i>M</i>. <i>tuberculosis</i> H37Rv mutant strains. Cells were grown in Middlebrook 7H9 liquid medium for 7 days with shaking. Intracellular glucans were measured in hot water extracts of washed cells. Capsular glucans were measured from cell-free culture supernatants. Intracellular and capsular glucans were assayed by sandwich ELISA employing an α-glucan specific monoclonal antibody. Similar results for intracellular glucan content were also obtained using an enzymatic assay with cells from independent biological replicates (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005768#ppat.1005768.s003" target="_blank">S3 Fig</a>). Values were normalized based on OD 600 nm of cultures. Values in (A) and (B) represent means of triplicates ± SEM. (C) Visualization of the α-glucan capsule in the <i>M</i>. <i>tuberculosis</i> WT and the Δ<i>glgC</i>(u) Δ<i>treS</i> mutant by immunogold labelling. Cells were grown in liquid medium without shaking, fixed, labelled with an α-glucan specific monoclonal antibody, and analyzed by electron microscopy (scale bar 0.5 μm). (D) Quantitative evaluation of α-glucan capsule visualization as shown in (C), plotted as anti-α-glucan specific gold particles per cell. Values represent means ± SEM (WT n = 27, Δ<i>glgC</i>(u) Δ<i>treS</i> n = 28). Negative controls were not treated with the primary anti-α-glucan antibody (n = 32).</p

Mycobacterial GlgA is an M1P synthesizing glucosyltransferase.

<p>(A) The preferred reaction catalyzed by GlgA from <i>M</i>. <i>tuberculosis</i>. (B) Recombinant <i>M</i>. <i>tuberculosis</i> GlgA converts ADP-glucose and G1P to M1P. ¹H NMR spectroscopy was used to monitor the anomeric protons of ADP-glucose and G1P (1 mM each) together with signals associated with the product in the presence of GlgA. The spectra show the concomitant consumption of ADP-glucose (~5.5 ppm) and appearance of resonances consistent with the formation of α-1,4 glycosidic linkages (~5.32 ppm). Given the lack of any resonances associated with free reducing ends (~5.1 ppm) and the retention of those associated with an α-glucosyl phosphate residue (~5.36 ppm), these observations are consistent with the formation of M1P. (C) Mass spectrometry of the product of the <i>M</i>. <i>tuberculosis</i> GlgA reaction with ADP-glucose and G1P. The dominant ion in the spectrum is consistent with the presence of M1P (<i>m/z</i> 421.0749 observed with 421.0752 expected for [M-H]^-). The next most abundant ion is consistent with the presence of the co-product, ADP (<i>m/z</i> 426.0218 observed with 426.0221 expected for [M-H]^-). (D) The dependence of <i>M</i>. <i>tuberculosis</i> GlgA activity on ADP-glucose. GlgA activity was determined spectrophotometrically by monitoring the production of ADP. A representative triplicate dataset with 1 mM G1P is shown as means and SEM. The data conform to the Michaelis-Menten <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005768#ppat.1005768.e001" target="_blank">eq 1</a> (fit shown as the solid line giving <i>r</i>² = 0.82). (E) The dependence of <i>M</i>. <i>tuberculosis</i> GlgA activity on G1P. GlgA activity was determined spectrophotometrically by monitoring the production of ADP. A representative triplicate dataset with 0.1 mM ADP-glucose is shown as means and SEM. Significant inhibition by G1P at concentrations >1 mM is apparent and the dataset conforms to a simple substrate inhibition <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005768#ppat.1005768.e002" target="_blank">eq 2</a> (fit shown as the solid line giving <i>r</i>² = 0.99). (F) <i>M</i>. <i>tuberculosis</i> GlgA (Rv1212c) was heterologously expressed in the <i>M</i>. <i>smegmatis</i> Δ<i>treS</i>(u) Δ<i>glgA</i>(u) c-<i>glgE</i>-tet-off mutant. Cells were cultivated for 24 h with or without 1 μg ml^-1 ATc as indicated, and hot water extracts from 1 ml culture aliquots (normalized to OD_{600 nm} = 0.5) were analyzed by TLC. Conditional silencing of the <i>glgE</i> gene results in M1P accumulation, demonstrating that <i>M</i>. <i>tuberculosis</i> GlgA synthesizes M1P <i>in vivo</i>.</p