Mycobacterium tuberculosis Deficient in PdtaS Cytosolic Histidine Kinase Displays Attenuated Growth and Affords Protective Efficacy against Aerosol M. tuberculosis Infection in Mice

Abstract

New control measures are urgently required to control tuberculosis (TB), as the current vaccine, Bacille Calmette-Guérin (BCG), has had a limited impact on disease spread. The identification of virulence mechanisms of Mycobacterium tuberculosis is an important strategy in vaccine design, as it permits the development of strains attenuated for growth that may have vaccine potential. In this report, we determined the role of the PdtaS response regulator in M. tuberculosis virulence and defined the vaccine potential of a pdtaS-deficient strain. Deletion of pdtaS (Mtb^ΔpdtaS) resulted in reduced persistence of M. tuberculosis within mouse organs, which was equivalent to the persistence of the BCG vaccine in the lung and liver of infected mice. However, the generation of effector CD4⁺ and CD8⁺ T cells (CD44⁺CD62L^loKLRG1⁺) was similar between wild-type M. tuberculosis and Mtb^ΔpdtaS and greater than that elicited by BCG. Heightened immunity induced by Mtb^ΔpdtaS compared to BCG was also observed by analysis of antigen-specific IFN-γ-secreting T cell responses induced by vaccination. Mtb^ΔpdtaS displayed improved protection against aerosol M. tuberculosis compared to BCG, which was most apparent in the lung at 20 weeks post-infection. These results suggest that the deletion of the PdtaS response regulator warrants further appraisal as a tool to combat TB in humans.

Keywords: PdtaS; immune response; tuberculosis; vaccine; virulence.

Figure 1

pdtaS^Tn growth is attenuated in macrophages and is protective as a vaccine against M. tuberculosis challenge. C57BL/6 mice (n = 4) were infected i.n. with 5 × 10⁵ CFU M. tuberculosis (WT Mtb), Mtb-mtrA^Tn, Mtb-pdtaS^Tn, or Mtb-pdtaR^Tn. Four weeks after infection, the CFU in the lungs (A) and spleen (B) was determined. RAW 264.7 cells were infected with BCG, WT Mtb, Mtb-mtrA^Tn, Mtb-pdtaS^Tn, or Mtb-pdtaR^Tn (MOI 1:1) for 4 h or 7 days at 37 °C, the intracellular bacterial load was determined (C), and fold change in CFU was calculated (D). C57BL/6 mice (n = 5) were vaccinated s.c. with 5 × 10⁵ CFU BCG, Mtb-pdtaS^Tn, or left unvaccinated. Ten weeks after vaccination, the mice were aerosol infected with M. tuberculosis H37Rv and 4 weeks after infection the M. tuberculosis CFU in the lungs (E) or spleens (F) was determined. The significances of differences between groups were determined by ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 2

Immune activation and bacterial persistence of Mtb^ΔpdtaS in mice C57BL/6 mice (n = 4) were infected s.c. with 5 × 10⁵ CFU M. tuberculosis (Mtb^WT), BCG, or M. tuberculosis deficient in pdtaS (Mtb^ΔpdtaS). Ten, thirty or seventy days post-vaccination, bacterial CFU in the lungs (A), spleen (B), or liver (C) was determined. The dotted line is the limit of detection. Representative flow cytometry plots show day 10 activated T cells (CD62L^-KLRG1⁺) after gating on CD4⁺CD44⁺ or CD8⁺CD44⁺ T cell populations (D). The total number of CD4⁺ (E) or CD8⁺ (F) activated T cells in the lungs is also shown. Results are representative of two independent experiments.

Figure 3

Antigen-specific IFN-γ production after vaccination with Mtb^ΔpdtaS. C57BL/6 mice were vaccinated, as in Figure 2. Ten, thirty or seventy days post-vaccination, IFN-γ production was assessed by ELISPOT in the spleen (A–C) and lung (D–F) after restimulation with culture filtrate protein (CFP). Results are representative of two independent experiments.

Figure 4

Protection afforded by Mtb^ΔpdtaS vaccination against M. tuberculosis aerosol challenge. C57BL/6 mice (n = 6) were vaccinated s.c. with 5 × 10⁵ CFU BCG or Mtb^ΔpdtaS. Ten weeks after vaccination, the mice were aerosol-infected with ~100 CFU M. tuberculosis, and 4 (A,C) or 20 (B,D) weeks after infection, the M. tuberculosis load in the lungs (A,B) or spleen (C,D) was determined. The percentage of CD4⁺ lung T cells expressing intracellular IFN-γ, IL-2 or TNF after restimulation ex vivo with CFP at weeks 4 (E) and week 20 (F) post-challenge is also shown. Results are representative of two independent experiments. The significances of differences between groups were determined by ANOVA (* p < 0.05, ** p < 0.01, and **** p < 0.0001; NS = not significant).