Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn-porphyrins

Abstract

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm²) with 565 nm light that correspond for Zn-porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

Figure 1

5-aminolevulenic acid (ALA) stimulates the synthesis of endogenous porphyrins in M. tuberculosis. Vegetative cells of M. tuberculosis cells were grown in Middlebrook 7H9 liquid medium supplemented with ADC and Tween-80 for 14 days (initial bacteria concentration 10⁵ cells per ml). After 5 days post inoculation 3 mM ALA was added to some flasks. Dormant Mtb cells were obtained in modified Sauton’s medium as described in “Materials and methods”. ALA (3 mM) was added to dormant culture after 20 days post inoculation. Porphyrin identification in chloroform–methanol extracts was carried out by LC–MS. Bars represented SD from two biological experiments.

Figure 2

Spectra of the extracts of vegetative and dormant M. tuberculosis cells grown in the presence of ALA. Mtb cells were inoculated in Middlebrook 7H9 liquid medium supplemented by ADC, Tween-80, and 3 mM ALA at a concentration of 10⁵ cells per ml, followed by incubation at 37 °C, under agitation of 200 rpm. After 20 days, biomass was collected by centrifugation and successively extracted with chloroform–methanol–water and then with 2% Triton × 100 (for details see the “Materials and methods”). Dormant Mtb cells were obtained as described in the “Materials and methods”. Then, 3 mM of ALA was added after 20 days post inoculation. Sixty-day-old dormant mycobacteria were harvested and porphyrins were extracted. The orange area corresponds to the illumination at 565/24 nm. (A) Absorption and fluorescence spectra chloroform–methanol-water extract of vegetative Mtb cells grown in the presence of ALA. Mostly free porphyrin was extracted with a certain amount of Zn porphyrin. (B) Absorption and fluorescence spectra at 410 nm excitation of 2% Triton X-100 extract of vegetative Mtb cells grown in the presence of ALA. Preferential extraction of Zn porphyrin with a small admixture of free porphyrin. (C) Absorption, fluorescence, and phosphorescence spectra at 410 nm excitation of 2% Triton × 100 extract of dormant Mtb cells grown in the presence of ALA. Preferential extraction of Zn porphyrin with a small admixture of free porphyrin. Experiments were repeated three times with similar results.

Figure 3

Single mycobacterial cell fluorescence emission spectra for both vegetative and dormant state. 20-day-old vegetative cells and 60-day-old dormant cells were applied to a glass cover slip and left to dry for a few minutes. Remaining cells were fixated with Merckoglass (Merck). Life-time images and fluorescence spectra of single bacteria were recorded by 405 nm laser excitation with 550 nm long-pass emission filter in confocal mode of measurements, (A)—life-time images for vegetative cells and (D)—for dormant cells with a magnified region of interest at the inserts, (B)—fluorescence spectra of porphyrins from single vegetative and (E)—dormant bacteria. Life-time decays for vegetative (C) and dormant (F) cells fit well by two exponential decay with Zn–porphyrin life-time (1.81 ns and 1.76 ns) and free base porphyrin (12.0 ns) correspondetly. Experiments were repeated five times for both vegetative and dormant cells.

Figure 4

HR-MS spectra in the negative ion mode of extracts of vegetative M. tuberculosis cells (A, B) and in the positive ion mode of extracts of dormant mycobacteria (C, D) in 75% acetone:15% methanol:10% water solution showing a characteristic isotopologue distribution of Zn–porphyrin complexes (A, C) and free base coproporphyrin (B, D), respective calculated distributions overlaid in red. Experiments were repeated three times with similar results.

Figure 5

Dependence of the change in the ratio of the level of gene expression in the comparison groups on the normalized average level of M. tuberculosis gene expression. The statistical significance of the changes (p value) was calculated based on the Wald statistic. Since multiple comparisons of experimental groups were used, the final value of p was calculated taking into account the Benjamini–Hochberg correction. (A) dormant cells versus active cells; (B) active cells treated with ALA versus active cells without ALA; (C) dormant cells treated with ALA versus dormant cells without ALA.

Figure 6

Photodynamic inactivation M. tuberculosis cells by light illumination at 565 nm. Dormant and vegetative Mtb cells were subjected to PDI as described in “Materials and methods”, at a different time of illumination at 565/24 nm (corresponding to the orange area in Fig. 4), under static conditions. After exposure, cell viability was estimated by MPN assay. Open triangles—vegetative Mtb, closed squares—dormant Mtb, closed triangles—vegetative Mtb grown in the presence of 3 mM ALA. Dormant mycobacteria had zero CFU. The experiments were repeated five times, and a representative result is shown. The MPN method was performed for two biological replicates. Three series of dilutions were made within each replic. Bars demonstrate (95%) confidence limits. Asterisks indicate that the results are significantly different from the control without ALA for every cell group by Student’s t-test. In the case of vegetative mycobacteria, data on the assessment of viable cells assessed by the CFU and MPN methods were close (Fig. S3B), in contrast to dormant forms. For unification, in all cases we used MPN assay. MPN method was performed for two biological replicates in every experiment.

Figure 7

Macrophage containing porphyrin-rich vegetative M. tuberculosis cell grown in presence of ALA. Vegetative Mtb cells can produce and accumulate porphyrins within 10 days of co-culture with lung macrophages in the presence of 1 mM ALA. In mycobacteria captured by macrophages, fluorescence characteristic of porphyrins was observed. Visualization of red-fluorescent mycobacteria (filter Lp590) within lung macrophage, counerstained with ActinGreen and DAPI (blue). The white arrow points to the mycobacteria inside the macrophage. Experiment was repeated two times with similar results.

Figure 8

Photodynamic inactivation of vegetative M. tuberculosis cells (A) and dormant M. tuberculosis cells (B, C) captured by lung macrophages. (A) After 10 days of co-culture vegetative mycobacteria with lung macrophages in the presence of 3 mM ALA. (B) Macrophages and dormant mycobacteria were grown separately. Dormant mycobacteria were obtained in the presence of 3 mM ALA. Macrophages were obtained in the presence and absence of 3 mM ALA. The bacteria were then captured by macrophages as described in the “Materials and methods”. (C) Macrophages and dormant mycobacteria were grown separately. Dormant mycobacteria were obtained in the absence of ALA. Macrophages were obtained in the presence and absence of 1 mM ALA. The bacteria were then captured by macrophages as described in the “Materials and methods”. (D) Fluorescence microscopy of macrophages grown in the presence and absence of ALA. Images were performed by invert fluorescent microscope Axio Observer.A1 (Zeiss, Germany), AxioCam MRc5, filter for porphyrins: BP 546/12—FT 580—LP 590. Photoinactivation was performed at 565 nm (300 J/cm²). Dormant mycobacteria and vegetative cells displayed zero CFU and 10⁷ CFU before illumination accordingly. Viability of the mycobacteria was estimated by MPN assay. MPN assessment was performed for two biological replicates in every experiment. Bars represent (95%) confidence limits. Experiments were repeated two times with similar results.