The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to nitric oxide

Abstract

Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (Delta prcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of Delta prcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function.

Figure 1. The proteasome is required for optimal growth in vitro and for resistance to RNI.

(A) Proteasome activities in H37Rv, ΔprcBA and the complemented mutant (ΔprcBA + PrcBA). The cleavage velocity (RFU/min) of the fluorogenic peptide substrate Suc-LLVY-AMC reports proteasome activity. Proteasome activity was not detectable in ΔprcBA. Data are means ± s.d. of three independent experiments. (B) Growth of H37Rv, ΔprcBA and ΔprcBA + PrcBA on agar plates. Serial dilution of the three stains were spotted onto 7H11 agar plates and incubated for 3 weeks at 37°C. (C) Growth of H37Rv, ΔprcBA and ΔprcBA + PrcBA in liquid culture. Strains were inoculated in 7H9 media and growth was followed by measuring OD₅₈₀. (D) Susceptibility to reactive nitrogen intermediates. Colony forming units (CFU) of H37Rv, ΔprcBA and the complemented strain after three days exposure to 0 mM and 3 mM sodium nitrite at pH 5.5. Data are means ± s.d. of triplicate cultures and representative of three independent experiments.

Figure 2. The proteasome is required for optimal growth and persistence in mice.

(A) Bacterial titers in lungs of C57BL/6 mice infected by aerosol with H37Rv, ΔprcBA and the complemented mutant. Data are means ± s.d. from four mice per time point per group and are representative of three independent experiments. (B) Gross pathology of lungs infected with H37Rv, ΔprcBA and complemented mutant at day 116 post-infection. (C) Bacterial titers in lungs of C57BL/6 mice infected by aerosol with H37Rv and ΔprcBA and treated with the iNOS inhibitor L-Nil and its inactive enantiomer D-Nil starting at day 25 post-infection (indicated by arrow). Data are means ± s.d. from four mice per time point per group and represent two independent experiments.

Figure 3. Characterization of the active site mutant proteasome.

(A) Proteasome activities of H37Rv, ΔprcBA and ΔprcBA complemented with the active site mutant proteasome (PrcAB-T1A) and the intact proteasome (PrcBA) measured using the fluorogenic substrate Suc-LLVY-AMC. Proteasome activity was not detectable in ΔprcBA and ΔprcBA complemented with the active site mutant proteasome (PrcAB-T1A). (B) PrcB levels analyzed by immunoblotting in lysates from H37Rv, ΔprcBA and the complemented strains. DlaT was used as loading control. (C) GFP activities in strains expressing GFP or the PanB-GFP fusion protein. (D) GFP and PanB-GFP levels analyzed by immunoblotting in lysates from H37Rv, ΔprcBA and the complemented strains. DlaT was used as loading control.

Figure 4. The active site mutant proteasome complements the growth defects and RNI hypersusceptibility of the proteasome KO.

(A) Growth of H37Rv, ΔprcBA and the complemented strains in liquid culture. Strains were inoculated in 7H9 media and growth was followed by measuring OD₅₈₀. (B). Growth of H37Rv, ΔprcBA and the complemented strains on agar plates. Serial dilutions of the indicated stains were spotted onto 7H11 agar plates and incubated for 3 weeks at 37°C. (C) Susceptibility to reactive nitrogen intermediates. Colony forming units (CFU) of H37Rv, ΔprcBA and the complemented strains after three days exposure to 0 mM, 3 mM or 5 mM sodium nitrite at pH 5.5. Data are means ± s.d. of triplicate cultures and representative of three independent experiments.

Figure 5. The active site mutant proteasome complements the in vivo growth defect but not the persistence defect of the proteasome KO.

(A) Bacterial titers in lungs of C57BL/6 mice infected by aerosol with ΔprcBA and ΔprcBA complemented with wild type and the active site T1A mutant ptroteasome. Data are means ± s.d. from four mice per time point per group and represent two independent experiments. (B) Gross pathology of lungs infected with ΔprcBA and ΔprcBA complemented with the wild type and the active site mutant proteasome at day 56 and day 120 post-infection. (C) Bacterial titers in lungs of C57BL/6 mice infected by aerosol with a mixed culture of H37Rv with a streptomycin resistance conferring plasmid integrated in the chromosomal attB site (H37Rv_strep) and the hygromycin resistant ΔprcBA.

Figure 6. A proteolytically intact core proteasome is required for long-term survival in stationary phase and during starvation.

(A) Growth and survival of H37Rv, ΔprcBA and the complemented strains in complete 7H9 growth medium. (B) Survival of H37Rv, ΔprcBA and the complemented strains in phosphate buffered saline. CFU were determined at indicated times by plating on 7H11 agar plates. Data are means ± s.d. of triplicates and representative of three independent experiments.