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. 2002 Jun 11;99(12):8330-5.
doi: 10.1073/pnas.102055799.

Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH

Deepak Kaushal  1 , Benjamin G Schroeder, Sandeep Tyagi, Tetsuyuki Yoshimatsu, Cherise Scott, Chiew Ko, Liane Carpenter, Jyoti Mehrotra, Yukari C Manabe, Robert D Fleischmann, William R Bishai
Affiliations

Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH

Deepak Kaushal et al. Proc Natl Acad Sci U S A. .

Abstract

The pathogenesis of tuberculosis involves multiple phases and is believed to involve both a carefully deployed series of adaptive bacterial virulence factors and inappropriate host immune responses that lead to tissue damage. A defined Mycobacterium tuberculosis mutant strain lacking the sigH-encoded transcription factor showed a distinctive infection phenotype. In resistant C57BL/6 mice, the mutant achieved high bacterial counts in lung and spleen that persisted in tissues in a pattern identical to those of wild-type bacteria. Despite a high bacterial burden, the mutant produced a blunted, delayed pulmonary inflammatory response, and recruited fewer CD4(+) and CD8(+) T cells to the lung in the early stages of infection. In susceptible C3H mice, the mutant again showed diminished immunopathology and was nonlethal at over 170 days after intravenous infection, in contrast to isogenic wild-type bacilli, which killed with a median time to death of 52 days. Complete genomic microarray analysis revealed that M. tuberculosis sigH may mediate the transcription of at least 31 genes directly and that it modulates the expression of about 150 others; the SigH regulon governs thioredoxin recycling and may be involved in the maintenance of intrabacterial reducing capacity. These data show that the M. tuberculosis sigH gene is dispensable for bacterial growth and survival within the host, but is required for the production of immunopathology and lethality. This phenotype demonstrates that beyond an ability to grow and persist within the host, M. tuberculosis has distinct virulence mechanisms that elicit deleterious host responses and progressive pulmonary disease.

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Figures

Figure 1
Figure 1
Lung (A) and spleen (B) cfu counts in C57BL/6 mice infected with wild-type M. tuberculosis (♦), the ΔsigH mutant (■), and the complemented strain (●) by the aerosol route. Groups of six mice infected were evaluated at each time point.
Figure 2
Figure 2
The ΔsigH mutant (B, E, H, and K) produces less tissue pathology in lungs of infected C57BL/6 and C3H mice compared with wild-type (A, D, G, and J) and complemented strains (C, F, I, and L). C57BL/6 mice infected by the aerosol route (as in Fig. 1) were evaluated at 16 weeks (A–C). C3H mice infected intravenously with 106 bacilli were analyzed at 8 weeks (D–F) and 16 weeks (G–I). In contrast, SCID mice infected intravenously with 104 bacilli and analyzed at 4 weeks (J–L) showed no significant difference in the degree of tuberculous pneumonia. (×40; hematoxylin and eosin staining.)
Figure 3
Figure 3
Flow cytometry of cell populations from the tissues of C57BL/6 mice infected with wild-type M. tuberculosis (♦) and the ΔsigH mutant (■) by the aerosol route. Each time point represents the mean cell count from a group of four mice. Absolute numbers of CD3+ cells from lungs (A) and spleens (B), and absolute numbers of lung CD4+ (C) and CD8+ (D) cells are shown. Intracellular cytokine staining for IFN-γ-positive CD4+ (E) and CD8+ (F) cells and for TNF-α-positive CD4+ (G) and CD8+ (H) cells from the lungs is also shown. Error bars represent one standard error.
Figure 4
Figure 4
Time-to-death analysis in C3H:HeJ (A) and SCID (B) mice upon intravenous. infection with M. tuberculosis wild-type strain (♦), the ΔsigH mutant (■), and the strain with complemented sigH gene (●) shows a significant mortality delay. The inoculum was 106 cfu for C3H mice (n = 10) and 104 cfu for SCID mice (n = 8).
Figure 5
Figure 5
(A) Putative sigH promoter consensus elements identified in 5′ untranslated regions of 31 genes with reduced expression in the ΔsigH mutant, and the corresponding fold-repression values and standard deviation (SD) from bacteria grown to OD600 = 1.2. Asterisks indicate that genes immediately downstream from these were also repressed and are probably part of an operon. Bases matching the consensus are shaded. (B) A consensus promoter recognition sequence for M. tuberculosis SigH derived from the 31 putative promoters, and the reported Streptomyces coelicolor SigR consensus (based on an analysis of three promoters) shown for comparison (40). Subscripts denote the percent frequency of occurrence of each base among the 31 genes shown. R = A or G.
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