Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening

Abstract

Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery, but subsequent target elucidation often proves difficult. Conditional mutants that underexpress essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA), and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility was demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery.

Figure 1. Growth of panC conditional mutants is highly ATc dose-dependent

(A) Growth on 7H10 agar. Strains were grown to mid-log phase and equivalent numbers of cells spotted on media containing the indicated concentrations of ATc and/or pantothenate. (B and C) Growth of panC Tet-ON_M and Tet-OFF in 7H9 liquid medium supplemented with the indicated concentrations of ATc and/or pantothenate. (D and E) Growth of the panC Tet-ON_M and panC Tet-OFF mutant strains relative to wt Mtb at the indicated ATc concentrations. Data are from a representative experiment performed in triplicate. See also Figure S3.

Figure 2. ATc-dependent PanC depletion in conditional mutant strains

(A) Western blot probed with PanC antibody (rabbit serum). Lanes were loaded with 25 ng of purified His-tagged PanC (A.K. & D.R.S., unpublished) (rPanC), molecular weight marker (Bio-Rad precision plus) (M), or 22.5 μg of whole cell lysates prepared from wild type H37RvMA (wt), panC SCO (SCO), and panC Tet-OFF or Tet-ON_M strains. Cells were grown in the presence or absence of 200 ng/ml ATc, as indicated. (B) The average level of PanC in each strain is shown as a percentage of the level in wt Mtb. The integrated signal intensity of each PanC band was quantified on a Li-Cor Infrared scanner in three replicate experiments performed with freshly prepared lysates (except panC-SCO which was performed in duplicate).

Figure 3. lysA conditional mutants show limited ATc dose-dependence for growth

(A) Growth on 7H10 agar. Strains were grown to mid-log phase and equivalent numbers of cells spotted on media containing the indicated concentrations of ATc and/or lysine. (B and C) ATc dose-dependent growth of lysA Tet-ON_M and Tet-OFF in 7H9 liquid medium containing ATc and/or lysine at the indicated concentrations. Data are from a representative experiment performed in triplicate.

Figure 4. Selective growth impairment of the icl1 Tet OFF strain in acetate

(A) Growth of icl1 Tet-OFF on 7H10 agar containing 0.1% acetate as the sole carbon source. The strain was grown to mid-log phase and equivalent numbers of cells spotted on media supplemented with the indicated concentrations of ATc. (B and C) Growth of the icl1 Tet-OFF conditional mutant in 7H9 media supplemented with 0.1% acetate (B) or glucose (C) in the absence (0 ATc) or presence (200 ATc) of inducer. Data are from a representative experiment performed in triplicate.

Figure 5. Target knockdown hypersensitizes Mtb to target-specific inhibitors

(A and B) icl1 silencing results in hypersensitivity to 3-NP. The susceptibility of wt Mtb, icl1-SCO and icl1 Tet-OFF to 3-NP inhibition was evaluated at the indicated concentrations of ATc during growth in media containing either glucose (A) or acetate (B) as the sole carbon source. Bacterial viability was measured by the Alamar Blue assay in which a transition from a blue to pink color is indicative of growth. (C and D) The susceptibility of the panC Tet-OFF strain to a representative PanC inhibitor, Alv553, (C) was evaluated (D) in 7H9 medium in the absence (−ATc) or presence (+ATc, 10 ng/ml) of ATc or presence of both ATc and pantothenate (+ATc + P). In each medium, the strain was treated with either the experimental compound (+) or the DMSO carrier alone (−). Bacterial growth, as determined by the quantity of bacterial cells settled at the bottom of each well, was assessed following incubation at 37° C for 7 days.

Figure 6. Fluorescence-based evaluation of the activity of target-specific inhibitors

(A) The susceptibility of panC Tet-OFF_GFP strain to a target-specific PanC inhibitor was assessed in 7H9 medium in the absence (−ATc) or presence (+ATc, 10 ng/ml) of ATc. Following incubation at 37° C for 7 days, the growth of the strain was measured by fluorescence quantification, and the RFU values normalized to growth levels without added drug. (B) The susceptibility of icl1 Tet-OFF_GFP strain to 3-NP inhibition was quantified in the presence (100 ng/ml) or absence of ATc during growth in media containing 0.1% acetate as the sole carbon source. Bacterial viability was measured by fluorescence measurement following incubation at 37° C for 7 days. RFU = relative fluorescence units. See also Figures S4 and S5.

Figure 7. qHTS of the panC Tet-OFF_GFP conditional mutant

(A) 3D scatter-plot of qHTS data either lacking (black) or possessing (blue) concentration–response curves. (B) Secondary screening of selected qHTS actives against the panC Tet-OFF_GFP strain in 7H9 medium in the absence (−ATc) or presence (+ATc, 50 ng/ml) of ATc or presence of ATc and pantothenate (+ATc/P).