Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients

Abstract

Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.

Fig. 1.

Sources of RNA samples. (A) MTB H37Rv was grown in Middlebrook 7H9 medium. Samples were withdrawn for OD₆₀₀ measurements and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR. •, OD₆₀₀; □, sigA/16S ratio × 10,000 ± SD (n ≥ 3). (B) C57BL/6 mice were aerosol-infected with ≈500 cfu of MTB H37Rv. Lungs were collected for cfu enumeration and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR. •, cfu ± SD (n ≥ 4); □, sigA/16S ratio × 10,000 ± SD (n ≥ 3). (C) Multilayered organization of granulomatous tissue bordering a 4-cm-diameter cavity in the left upper lung lobe of patient 2 (hematoxylin/eosin-stained). At the periphery were residual airspace and lymphocytic aggregates (i). A capsule of fibroblasts and fibrin with scattered epithelioid macrophages (ii) surrounded a cellular zone containing clusters of multinucleated giant cells (iii). Giant cells were also prominent at the border between the cellular zone and the underlying zone of caseation necrosis (iv). The surface of the cavity wall bordering on the necrotic zone showed extensive leukocytic infiltration and numerous acid-fast bacilli, not revealed by hematoxylin/eosin staining (v). Magnification: ×4(Left), ×40 (Right). Similar pathology was observed in lung specimens from all four patients analyzed in this study.

Fig. 2.

MbtB peptide synthetase (mbtB) mRNA levels in MTB grown in vitro (A and B; gray bars), in aerosol-infected mice (A and B; black bars), and in lung specimens from TB patients (C). Ratios of mbtB/16S (A) or mbtB/sigA (B and C) are means ± SD (n ≥ 3).

Fig. 3.

Bacterioferritin A (bfrA) mRNA levels in MTB grown in vitro (A and B; gray bars), in aerosol-infected mice (A and B; black bars), and in lung specimens from TB patients (C). Ratios of bfrA/16S (A) or bfrA/sigA (B and C) are means ± SD (n ≥ 3).

Fig. 5.

Malate synthase (mls) mRNA levels in MTB grown in vitro (A and B; gray bars) and in aerosol-infected mice (A and B; black bars). Ratios of mls/16S (A) or mls/sigA (B) are means ± SD (n ≥ 3).

Fig. 7.

Pyruvate carboxylase (pca) mRNA levels in MTB grown in vitro (A and B; gray bars) and in aerosol-infected mice (A and B; black bars). Ratios of pca/16S (A) or pca/sigA (B) are means ± SD (n ≥ 3).

Fig. 4.

Isocitrate lyase (icl1) mRNA levels in MTB grown in vitro (A and B; gray bars), in aerosol-infected mice (A and B; black bars), and in lung specimens from TB patients (C). Ratios of icl1/16S (A) or icl1/sigA (B and C) are means ± SD (n ≥ 3).

Fig. 6.

pckA mRNA levels in MTB grown in vitro (A and B; gray bars), in aerosol-infected mice (A and B; black bars), and in lung specimens from TB patients (C). Ratios of pckA/16S (A) or pckA/sigA (B and C) are means ± SD (n ≥ 3).

Fig. 8.

α-crystallin (hspX) mRNA levels in MTB grown in vitro (A and B; gray bars), in aerosol-infected mice (A and B; black bars), and in lung specimens from TB patients (C). Ratios of hspX/16S (A) or hspX/sigA (B and C) are means ± SD (n ≥ 3). hspX mRNA could not be quantified in lung specimen 3.1 from patient 3 (C) because of a polymorphism in hspX in this patient's strain that interfered with QRT-PCR.