Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages

Abstract

Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.

FIG. 1.

M. tuberculosis 16S rRNA:DNA ratio in the mouse lung and in broth culture. RNA and genomic DNA were simultaneously isolated from M. tuberculosis growing in aerated broth culture (mid-log phase) and in lungs of infected mice at 2, 4, and 12 weeks postinfection. 16S rRNA and DNA copy numbers were quantified with qPCR, and the log copy number of 16S rRNA was regressed on DNA for data from in vitro and in vivo bacteria. Three models were compared by multiple regression analysis (SPSS version 12): (i) a model with two separate slopes and intercepts, (ii) a model with parallel lines but differing intercepts, and (iii) a single line for both data sets. Comparison of the three models indicated that the regression lines of in vitro and in vivo data did not differ statistically either in slope (0.9025 and 0.9736) or intercept (3.4815 and 3.3362) at an alpha of 0.05, and thus the third model was accepted to best describe the data. Four mice were included per time point. Data are representative of two independent experiments. wk, week.

FIG. 2.

Regulation of M. tuberculosis whiB3 and acr in wild-type C57BL/6 and immunodeficient mice. RNA was isolated from M. tuberculosis in lungs of infected mice at the indicated time points. Quantitative RT-PCR was performed for whiB3 (a) and acr (b) and normalized to 16S rRNA in the same sample. Fold induction, defined as a ratio of normalized expression in mouse lung to mid-log broth culture, was calculated. The inset shows CFU equivalents calculated based on the 16S rRNA copy numbers. TNF-α^−/− mice did not survive beyond week 4. Bars show mean induction plus standard deviations of quadruplicates. Wild-type and IFN-γR^−/− data are representative of three independent experiments. The difference in whiB3 induction at 2 weeks was not reproducible. *, P < 0.05 compared to same time point in the wild-type; **, P < 0.0001 compared to same time point in the wild-type. wk, week; WT, wild type; eq., equivalent.

FIG. 3.

Regulation of M. tuberculosis whiB3 in naïve and IFN-γ-activated macrophages. Induction of whiB3 in resting (a) and activated (b) BMMφ after infection with an MOI of 5:1. Quantitative RT-PCR was performed and normalized to 16S rRNA from the same sample. Fold induction is defined as the ratio of normalized expression in BMMφ to broth culture. Bars show means plus standard deviations of biological triplicates. icl and Rv1200 represent positive and negative controls, respectively. icl induction in naïve macrophages is consistent with that reported previously (23). Data are representative of two to four independent experiments. *, P < 0.05 compared to untreated macrophages; **, P < 0.005 compared to untreated macrophages.

FIG. 4.

Correlation of whiB3 expression with bacterial density. Correlation curve for the expression of whiB3 and CFU equivalents in bacteria from the lungs of wild-type (WT) and immunodeficient mice at 2, 3, 4, and 5 weeks postinfection (a) and resting BMMφ infected with MOIs ranging from 1:6 to 8:1 (b). (c) Expression of whiB3 at the indicated bacterial densities in broth culture for 10 h. Bars show means plus standard deviations. Data are representative of two to three independent experiments done in quadruplicates (a), triplicates (b), and duplicates (c). WT, wild type; eq., equivalent.

FIG. 5.

Disruption of M. tuberculosis whiB3. (a) Genomic map of whiB3 and flanking genes in M. tuberculosis H37Rv. (b) AES used to make a whiB3 knockout mutant. The shaded box with a hyg cassette represents the region deleted from whiB3. (c) Genomic fragment PCR amplified to create a ΔwhiB3attB::whiB3 complement. (d) Phenotypic confirmation of ΔwhiB3 by real-time RT-PCR. The expression of whiB3 transcript in H37Rv, ΔwhiB3 (whiB3 mutant), and ΔwhiB3attB::whiB3 (whiB3 complement) is shown.

FIG. 6.

Phenotype of whiB3 knockout in wild-type and IFN-γR^−/− mice. (a) Bacterial counts from the lungs of C57BL/6 mice infected with H37Rv, whiB3 knockout (ΔwhiB3), and the complement (+whiB3). Bars show means plus standard deviations of four mice on day 1 and two mice on day 154. (b and c) Images of lungs and sections taken from C57BL/6 mice infected with H37Rv (Rv) and ΔwhiB3 at 154 days postinfection. Magnification, ×40. (d) Survival of IFN-γR^−/− mice (13 per group) infected with aerosolized M. tuberculosis. Day 1 CFU counts were 100 and 65 for H37Rv and ΔwhiB3, respectively.