DosS Is required for the complete virulence of mycobacterium tuberculosis in mice with classical granulomatous lesions

Abstract

Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.

Keywords: Mycobacterium tuberculosis; hypoxia; latency; response regulator; sensor kinase.

Figure 1.

Determination of bacterial burden in vivo. Levels of CFUs were determined in the tissues of infected animals at Day 1 and every 4 weeks thereafter. A comparable low-dose infection was established in the lungs of each of the C3HeB/FeJ mice used. Mtb (red); ∆-dosR (black); ∆-dosS (blue); and ∆-dosT (green). (A) CFUs in lungs. (B) CFUs in liver. Results are expressed as CFUs in the entire tissue based on weight. *P < 0.05; **P < 0.005 (unpaired t test). Mtb, Mycobacterium tuberculosis.

Figure 2.

Immunohistochemistry to localize bacilli in the lungs of C3HeB/FeJ mice. (A) Diaminobenzidine-based detection of bacilli (brown color) in mice lungs infected with Mtb or Δ-dosS. Two representative images from 10 fields per slide out of a total of three slides per animal are shown. Multiple cells staining positive for bacilli counts are shown on the corresponding higher magnification image to the right of each panel. (B) Multilabel confocal microscopy specifically shows the presence of intracellular bacilli (green signal) in the lungs of mice infected with Mtb (left) and the Δ-dosS mutant (right) or at early time points (e.g., 8 wk after infection) (top panel). Lesions with central necrosis were observed for both infection groups at later time points (e.g., Week 20). Here, bacilli were mostly extracellular (bottom panels). Scale bars: A, 100 μm in first and third columns, 50 μm in second and fourth columns; B, 22 μm.

Figure 3.

Histopathology analysis. Using hematoxylin and eosin staining, we determined the extent of histopathology in the lungs of animals infected with the different strains over the course of time. (A) Day 1. Non-necrotic lung lesions in the early stages of development are composed primarily of lymphocytes and macrophages. (B) Week 12. Lesions accumulate neutrophils, foamy macrophages, and numerous cholesterol clefts. (C) Percentage of lung area grouped from lung lobes of four mice in Mtb- (red) and Mtb:∆-dosS- (blue) infected mice at 4 and 12 weeks after infection. Results show a significant difference at the latter time point. *P = 0.0330; **P = 0.0066 (unpaired t test).

Figure 4.

Disease progression in C3HeB/FeJ mice lungs infected with Mtb strains. (A and B, upper panels) At Week 16, lesions have individual foamy macrophage (blue arrow) with intracellular (green arrow) and extracellular (black arrow) cholesterol clefts (upper panel) stained with Oil Red O, a lipid-staining dye (red arrow, right upper panels in A and B) in lungs of mice infected with Mtb (A) or Δ-dosS (B). (A and B, lower panels) At Week 20, lesions with necrosis or approaching being nearly necrotic are shown. Stain: hematoxylin and eosin (H&E). N indicates areas of necrosis.

Figure 5.

Supervised hierarchical clustering and global gene-ontology analysis. (A) Expression of chemokines, interferons/co-regulated genes, and cytokines/TNF c-regulated genes in Mtb- and Δ-dosS-infected lungs at Week 8. The intensity of red color correlates with higher levels of induction; the intensity of blue color correlates with higher levels of repression relative to the expression of the same genes in Day 1 (baseline) animals. Bar represents the range of gene-expression magnitude. (B) The relative fold change in transcripts (Mtb-infected mice Week 8 to Mtb-infected mice, Day 1) or (∆-dosS-infected mice, Week 8, to ∆-dosS-infected mice, Day 1) in microarray (red or blue bars) and quantitative RT-PCR (dark red or dark blue bars) is shown.

Figure 6.

Detection of hypoxia in vivo and evaluation of the in vitro hypoxia phenotype of the Mtb:Δ-dosS mutant. (A) Immunohistochemistry staining with hypoxia marker pimonidazole in lungs of C3HeB/FeJ mice infected with Mtb (left) and the Mtb:∆-dosS mutant (right). Multiple cells stain positive in each of the two images, and the highest intensity is reserved in the inner-ring wall of each of the lesions, where presumably cells are intact, relative to the central necrotic region. (B) Quantitative RT-PCR–based evaluation of expression of the various members of the DosR regulon (dosR, dosS, dosT, Rv1738, hspX, tgs1, and Rv3131 [39], the various strains grown in hypoxic conditions in vitro). Mtb (red); Δ-dosR (white); Δ-dosS (blue); and Δ-dosT (green). The y-axis represents fold change of expression relative to when these strains were cultured in normal aerobic conditions. Results were normalized based on the C_t values for the Mtb 16S ribosomal RNA gene as a housekeeping control. (C) CFUs per milliliter of Mtb, Δ-dosR, Δ-dosS, and Δ-dosT grown in aerobic (Aer) conditions (solid bar) and in hypoxic (Hyp) conditions (35-d standing cultures) (open bar) are shown. Each bar represents results from biological replicate experiments.

Figure 7.

Growth–phenotype comparison of Mtb:Δ-dosS to Mtb C3HeB/FeJ mouse–derived bone marrow–derived macrophages (BMDMs). (A) Growth of Mtb:Δ-dosS was compared with wild-type Mtb in IFN-γ–activated C3HeB/FeJ mouse–derived BMDMs. Mtb (red); Δ-dosS (blue). Bacterial counts were significantly different at and after 24 hours (P < 0.0001; unpaired t test). Each bar represents results from biological replicate experiments. (B) Fold change in regulation of selected genes by quantitative RT-PCR, microarray (C) in BMDMs infected with Δ-dosS relative Mtb. ***P < 0.005. ns, not significant.