Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence

Abstract

The lung is the primary target of infection with Mycobacterium tuberculosis. It is well established that, in mouse lung, expression of adaptive, Th1-mediated host immunity inhibits further multiplication of M. tuberculosis. Here, real-time RT-PCR was used to define the pattern of expression against time of lung infection of key genes involved in Th1-mediated immunity and of selected genes of M. tuberculosis. Inhibition of bacterial multiplication was preceded by increased mRNA synthesis for IFN-gamma and inducible NO synthase (NOS2) and by NOS2 protein synthesis in infected macrophages. Concurrently, the pattern of transcription of bacterial genes underwent dramatic changes. mRNA synthesis increased for alpha-crystallin (acr), rv2626c, and rv2623 and decreased for superoxide dismutase C (sodC), sodA, and fibronectin-binding protein B (fbpB). This pattern of M. tuberculosis transcription is characteristic of the nonreplicating persistence [Wayne, L. G. & Sohaskey, C. D. (2001) Annu. Rev. Microbiol. 55, 139-163] associated with adaptation of tubercle bacilli to hypoxia in vitro. Based on this similarity, we infer that host immunity induces bacterial growth arrest. In IFN-gamma gene-deleted mice, bacterial growth was not controlled; NOS2 protein was not detected in macrophages; sodC, sodA, and fbpB transcription showed no decrease; and acr, rv2626c, and rv2623 transcription increased only at the terminal stages of lung pathology. These findings define the transcription signature of M. tuberculosis as it transitions from growth to persistence in the mouse lung. The bacterial transcription changes measured at onset of Th1-mediated immunity are likely induced, directly or indirectly, by nitric oxide generated by infected macrophages.

Figure 1

IFN-γ and NOS2 mRNA synthesis during the course of M. tuberculosis lung infection. WT C57BL/6 mice were infected with 2 × 10² CFU of M. tuberculosis H₃₇Rv via the respiratory route. At the times indicated, bacterial CFU in the mouse lung were determined. Total RNA was isolated from lung, and the copy numbers per lung of IFN-γ and NOS2 mRNA were measured by real-time RT-PCR, as described in Materials and Methods. (A) Course of M. tuberculosis infection in mouse lung. Each data point represents the mean (±SD) of M. tuberculosis CFU (in log units) obtained from five mice per time point. (B and C) Measurements of copy numbers of mRNA of IFN-γ (B) and NOS2 (C). mRNA copy number (log units per lung) were determined in triplicate by using pooled RNA from the lungs of three mice killed at the times indicated. Shown are the means (±SD) of three separate determinations of the pooled RNA. In separate, ongoing experiments, we have determined that the number of IFN-γ and NOS2 mRNA copies is essentially the same immediately before (day −1) and immediately after (day +1) aerosol infection with M. tuberculosis (data not shown). These basal levels are explained by the continued exposure of the lung to environmental agents, including living microorganisms.

Figure 2

Sections of lungs of M. tuberculosis-infected WT and IFN-γ^−/− mice stained for acid-fast bacteria and for NOS2 by immunocytochemistry. On day 20 of infection, lung lesions in WT mice (a) consisted of accumulation of macrophages that stained positively for NOS2 (brown color) and contained acid-fast bacteria (red rods). In contrast, macrophages containing acid-fast bacilli in lesions of IFN-γ^−/− mice (b) failed to stain for NOS2. On day 35 of infection, lung lesions in WT mice (c) were of similar appearance to those on day 20. However, day-35 lung lesions in IFN-γ^−/− mice (d) were more numerous and much larger than those in WT mice (not shown) and were clearly of a different cellular composition, being populated predominantly by neutrophils, most of which were dead and degenerating, and many of which contained acid-fast bacilli. (Magnification, ×650.)

Figure 3

Correlation of CFU with the copy number of 16S rRNA from M. tuberculosis growing in the lungs of WT and IFN-γ^−/− mice. Lungs were harvested from M. tuberculosis-infected mice at selected times during infection. (A) Half the lung (attached to the right bronchus) was used to quantitate the number of copies of 16S rRNA by molecular-beacon real-time RT-PCR, and the other half (attached to the left bronchus) was used to determine the number of CFU. The data points shown in the figure represent mean (±SD) of data (expressed in log units) per lung obtained from four animals per time point per mouse strain. (B) Correlation curve for CFU and 16S rRNA copies (in log units) obtained with lungs from WT and IFN-γ^−/− mice pooled together. Correlation coefficients obtained separately with WT mice (R² = 0.9536) and with IFN-γ^−/− mice (R² = 0.9570) were similar to that shown in B.

Figure 4

Quantitation by molecular-beacon real-time RT-PCR of selected mRNAs of M. tuberculosis in the lungs of WT and IFN-γ^−/− mice during the course of infection. Lungs were harvested from M. tuberculosis-infected mice at selected times during infection. RNA isolated from the half lung used for 16S rRNA measurements (see Fig. 3) was also used to determine the number of copies of selected mRNAs by molecular-beacon real-time RT-PCR. Shown are the means ± SD (in log units) of data obtained for two bacterial genes, acr and sodC, by using lungs from four mice per time point per mouse strain. Raw data for four additional M. tuberculosis genes are presented in the supporting information on the PNAS web site.

Figure 5

Normalized copy numbers of mRNA of M. tuberculosis acr (A), rv2626 (B), and rv2623c (C) in the lungs of WT and IFN-γ^−/− mice during the course of infection. The number of copies of M. tuberculosis mRNA per lung was measured by molecular-beacon real-time RT-PCR as described in Materials and Methods and in the legend to Fig. 4. At each time point and for each mouse strain, normalized mRNA values were obtained by dividing the mean of mRNA copy numbers per lung (shown for acr in Fig. 4 and for the remaining genes in the supporting information on the PNAS web site) by the corresponding mean of 16S RNA copy numbers per lung (Fig. 3A). Shown are the ratios of mRNA and 16S rRNA. Each panel presents results obtained with one gene, as indicated.

Figure 6

Normalized copy numbers of sodC, sodA, and fbpB mRNA of M. tuberculosis in the lungs of WT and IFN-γ^−/− mice during the course of infection. Normalized mRNA values for sodC (A), sodA (B), and fbpB (C) were calculated as described in the legend to Fig. 5.