mosR, a novel transcriptional regulator of hypoxia and virulence in Mycobacterium tuberculosis

Abstract

Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the DeltamosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.

FIG. 1.

Deletion, complementation and overexpression of Rv0348. (A) Organization of the Rv0348 operon and our strategy for gene disruption. See Materials and Methods for details. (B) Southern blot analysis of SalI-digested genomic DNA of the H37Rv wild type and the ΔmosR mutant. (C) PCR analysis of cDNA synthesized from RNA samples purified from H37Rv (lanes 1, 3, and 5) or the ΔmosR mutant (lanes 2, 4, and 6). (D) Western blot analysis of different M. tuberculosis strains using polyclonal antibodies raised in rabbits against the MBP-MosR protein. Pellets from M. tuberculosis H37Rv (lane 1), the ΔmosR mutant (lane 2), the ΔmosR::mosR complemented strain (lane 3), and the H37Rv::mosR overexpression strain (lane 4) were subjected to immunoblotting. The blot for soluble fractions was negative (data not shown).

FIG. 2.

Transcriptional profile of mosR in M. tuberculosis H37Rv under various stressors. Shown is qRT-PCR of mosR transcripts under various stress conditions, such as high temperature (45°C) (Temp), H₂O₂ (10 mM), and SDS (0.05%) treatments, as well as transition to stationary phase (OD₆₀₀ = 1.5). The change was calculated relative to transcripts in untreated cultures of M. tuberculosis strain H37Rv (OD₆₀₀ = 0.5). The error bars represent standard deviations from the means.

FIG. 3.

Comparative analysis of the transcriptome of M. tuberculosis exposed to various conditions. (A) Venn diagram representing the number of positively mosR-regulated genes compared to genes induced under nutrient starvation (3) and those repressed in the phagosome environment (36). (B) Venn diagram representing the number of negatively mosR-regulated genes compared to genes induced under hypoxia (27) and anaerobic conditions (25).

FIG. 4.

Transcriptional activity of the mosR operon. (A) EMSA of MosR with different promoter regions of significantly regulated genes as detected by DNA microarrays. EMSA was performed as described previously (44). Gene names are listed above the lanes. (B) Western blot analysis of the recombinant strain of M. smegmatis mc²155 expressing MosR protein. (C) β-Galactosidase activities of constructs for Rv3130c (tgs1), Rv0347, Rv0700, and Rv0167 promoters fused to the coding sequence of lacZ in the presence/absence of the mosR operon using M. smegmatis cells as host cells. The asterisks denote significant changes in a Student t test (P < 0.001). The error bars indicate standard deviations.

FIG. 5.

A regulatory role for mosR under anaerobic conditions. (A) The survival curve of M. smegmatis::pML21 under aerobic and anaerobic conditions (left scale) and the change in mosR transcripts for hypoxic versus aerobic cultures as measured by qRT-PCR (right scale). The error bars indicate standard deviations. (B) Experimental design for the regulation of the tgs1 gene under the control of mosR. Cultures of recombinant M. smegmatis (rM. smegmatis) harboring the mosR operon (OpmosR) were grown under anaerobic conditions using the Wayne model of hypoxia. Samples were collected at different times following incubation based on fading of the methylene blue color and transition from aerobic (dark blue) to anaerobic (colorless) stages. gDNA, genomic DNA. (C) β-Galactosidase activities of anaerobic cultures of recombinant M. smegmatis harboring pML28 with or without the mosR operon. All activities were normalized to the total protein content in each sample. The asterisks denote significant changes in a Student t test (P < 0.001).

FIG. 6.

Colonization of M. tuberculosis with various copies of mosR. (A) Lung colonization levels following aerosol infection with H37Rv, ΔmosR, ΔmosR::mosR, and Δcso strains. The asterisks denote significant differences in these times (Student's t test). The error bars indicate standard deviations. GM, gram. (B) Survival curves of mouse groups (n = 10) following aerosol infection with various strains of M. tuberculosis. The Δcso mutant was included as an example of a high inducer of PDIM.

FIG. 7.

Lesions associated with infection with M. tuberculosis with different copies of mosR. Shown is a histological analysis of sections of mouse lungs at 2 and 20 weeks (WKS) postinfection with the ΔmosR, ΔmosR::mosR, and H37Rv strains. Note the levels of accumulation of inflammatory cells (arrows) in hematoxylin- and eosin-stained sections. Aggregates of macrophages and lymphocytes (arrowheads) are displayed inside insets at higher magnification. Bars, 100 μm or 200 μm.

FIG. 8.

MALDI-TOF mass spectra of alkali-treated lipids from M. tuberculosis H37Rv, ΔmosR, ΔmosR::mosR, and Δcso strains. The values indicate the masses of the major pseudomolecular ions [M + Na]⁺ of MAMEs and PDIM as indicated above the spectra. Lipid samples were dissolved in chloroform and applied to the target plate. 2,5-dihydroxy benzoic acid was used as a matrix (10 mg/ml in CHCl₃/CH₃OH; 1:1 [vol/vol]).