The role of RelMtb-mediated adaptation to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice

Abstract

Long-term survival of nonreplicating Mycobacterium tuberculosis (Mtb) is ensured by the coordinated shutdown of active metabolism through a broad transcriptional program called the stringent response. In Mtb, this response is initiated by the enzymatic action of RelMtb and deletion of relMtb produces a strain (H37RvDeltarelMtb) severely compromised in the maintenance of long-term viability. Although aerosol inoculation of mice with H37RvDeltarelMtb results in normal initial bacterial growth and containment, the ability of this strain to sustain chronic infection is severely impaired. Significant histopathologic differences were noted in lungs and spleens of mice infected with H37RvDeltarelMtb compared with controls throughout the course of the infection. Microarray analysis revealed that H37RvDeltarelMtb suffers from a generalized alteration of the transcriptional apparatus, as well as specific changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat shock proteins, and secreted antigens that may alter immune recognition of the recombinant organism. Thus, RelMtb is critical for the successful establishment of persistent infection in mice by altering the expression of antigenic and enzymatic factors that may contribute to successful latent infection.

Fig. 1.

Growth of Rel_MTb-deficient H37Rv in the lungs (a) and spleens (b) of low-dose aerosol-infected C57BL/6 mice. In both graphs, open squares represent the colony-forming units (cfu) of wild-type H37Rv and open triangles represent the cfu of the RelMtb-defective strain H37RvΔrel_Mtb. Filled triangles represent the complemented strain H37RvΔrel_MtbattB::rel_Mtb. These data are representative of three independent experiments.

Fig. 2.

Weight gain (a) and gross pathology in lungs (b) and spleens (c) of aerosol-infected C57BL/6 mice. (a) Mean ± SD for the weights of groups of four mice evaluated 15 weeks after infection. (b) The lungs of mice infected with wild-type H37Rv (Top), H37RvΔrel_Mtb (Middle), and H37RvΔrel_MtbattB::rel_Mtb (Bottom). (c) Spleens of mice infected with wild-type H37Rv (left), H37RvΔrel_Mtb (center), and H37RvΔrel_MtbattB::rel_Mtb (right).

Fig. 3.

Histopathology of the lungs of aerosol-infected mice 15 weeks after exposure. (a, d, and g) Wild-type H37Rv. (b, e, h) H37RvΔrel_Mtb. (c, f, and i) H37RvΔrel_MtbattB::rel_Mtb. (a–c) ×4 magnification. (d–f) Granulomatous tissue at ×40 magnification. (g–i) Uninvolved lung tissue at ×40 magnification.

Fig. 4.

Histopathology of the lungs of aerosol-infected mice 38 weeks after infection. (a and d) Wild-type H37Rv. (b and e) H37RvΔrel_Mtb. (c and f) H37RvΔrel_MtbattB::rel_Mtb. (a–c) ×4 magnification. (d–f) Granulomatous tissue at ×40 magnification.