In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

Abstract

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-gamma-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.

Figure 1. Tet-ON and Tet-OFF systems allow efficient and rapid silencing of proteasome expression

(a) Map of the prcBA genomic region in H37Rv wild type (top) and in H37Rv P_myc1tetO:prcBA (bottom). Probe location and EcoRV restriction sites are indicated. (b) Southern blot of EcoRV digested genomic DNA from wild type H37Rv and from three transductants of H37Rv P_myc1tetO:prcBA (t1, t2, t3) probed with the DNA fragment indicated in (a). (c) PrcB levels analyzed by immunoblot in H37Rv P_myc1tetO:prcBA without TetR and transformed with wtTetR (PrcBA_Tet-ON, left panel) and with revTetR (PrcBA_Tet-OFF, right panel) after growth for 7 days in the presence and absence of anhydrotetracycline (atc). Dihydrolipoamide acyltransferase (DlaT) was used as loading control. (d) PrcBA transcript levels after transformation of H37Rv P_myc1tetO:prcBA with wtTetR (PrcBA_Tet-ON, left panel) and with revTetR (PrcBA_Tet-OFF, right panel) and growth for 7 days in the presence of atc (white bars) and in the absence of atc (black bars). H37Rv P_myc1tetO:prcBA not transformed with TetR (no TetR) was included as control; normalization of prcBA transcript to rpoB and 16S rRNA instead of sigA gave similar results (data not shown); data are means ± s.e. of triplicate samples. (e) Proteasome activities in PrcBA_Tet-ON (circles, left panel) and PrcBA_Tet-OFF (diamonds, right panel) mutants in presence (open symbols) and absence (filled symbols) of atc; nd = not detected. (f) PrcB levels in PrcBA_Tet-ON (left panel) and PrcBA_Tet-OFF (right panel) mutants in the presence and absence of atc at indicated time points. DlaT was used as loading control.

Figure 2. In vitro phenotypes caused by prcBA silencing

(a) Growth in liquid culture determined by optical density and on agar plates (insets) of PrcBA_Tet-ON (left panel) and PrcBA_Tet-OFF (middle panel) mutants and wild type Mtb (H37Rv, no TetR, right panel) in the presence (open symbols, +) and absence (filled symbols, −) of anhydrotetracycline (atc). (b) Susceptibility of PrcBA_Tet-ON and PrcBA_Tet-OFF mutants to RNI and H₂O₂. The indicated strains were grown for 7 days in the presence or absence of atc and then exposed to 3 mM NaNO₂ at pH 5.5 for 3 days and 5 mM H₂O₂ at pH 6.8 for 4 hours at 37°C in the presence (white bars) and absence (black bars) of atc and enumerated by plating for colony-forming-units (cfu); data are means ± s.e. of 2 independent experiments each with triplicate cultures (exposure to NaNO₂) or 1 experiment representative of 2 with triplicate cultures (exposure to H₂O₂).

Figure 3. Induction of GFP expression in Mtb in mouse lungs

(a) Lung sections from mice infected with Mtb transformed with P_myc1tetO-gfp (upper panels) and P_myc1tetO-gfp/wtTetR (GFP_Tet-ON, middle and lower panels). Two weeks post infection a group of mice (lower panels) received doxycycline (doxy) for 6 days in the drinking water while the control groups were kept without doxy (middle panels). The left panels depict lung tissues after rhodamine-auramine staining to detect Mtb (magnification: 60×). The middle and right panels show unstained fluorescent images obtained with a GFP filter at two magnifications (60× and 150×). (b) Bacterial titres in lungs of mice infected with P_myc1tetO-GFP (green bars) and P_myc1tetO/wtTetR (GFP_Tet-ON). Cfu from mice treated with doxy starting 2 days and 14 days post infection are depicted as yellow and orange bars, respectively. Control groups were kept without doxy (green and black bars). Data are means ± s.d. from 4 mice per group and time point. Cfu counts from mice treated with doxy at day 12 were not significantly different from the other two data points according to a Students t-test.

Figure 4. The proteasome is essential for optimal growth and persistence of Mtb in mice

(a) Mice were infected by aerosol with the PrcBA_Tet-ON mutant and administered doxycycline (doxy, green squares) in the drinking water or given regular water (blue diamonds). Bacterial loads in lungs were enumerated by plating for cfu at indicated time points. (b, c, d, e) Mice were infected by aerosol with (b, c) the PrcBA_Tet-OFF mutant and (d, e) wild type H37Rv transformed with revTetR. Mice were given doxy starting on day 7 (green squares, bars) and day 21 (orange triangles, bars) or given regular water (blue diamonds, bars). Bacterial loads in lungs (b, d) and spleens (c, e) were enumerated by plating for cfu at indicated time points. Data are means ± s.d. of 4 mice per group and time point and are representative of 2 independent experiments. (f) Survival of IFNγ^−/− mice infected by aerosol with the PrcBA_Tet-OFF mutant. At day 10 post infection half (n=10) of the infected mice received doxy in their drinking water (green squares) the other half received regular water (blue diamonds).