Depletion of antibiotic targets has widely varying effects on growth

Abstract

It is often assumed that antibiotics act on the most vulnerable cellular targets, particularly those that require limited inhibition to block growth. To evaluate this assumption, we developed a genetic method that can inducibly deplete targeted proteins and that mimics their chemical inactivation. We applied this system to current antibiotic targets in mycobacteria. Although depleting some antibiotic targets significantly perturbs bacterial growth, surprisingly, we found that reducing the levels of other targets by more than 97% had little or no effect on growth. For one of these targets, dihydrofolate reductase, metabolic analysis suggested that depletion mimics the use of subinhibitory concentrations of the antibiotic trimethroprim. These observations indicate that some drug targets can exist at levels much higher than are needed to support growth. However, protein depletion can be used to identify promising drug targets that are particularly vulnerable to inhibition.

Fig. 1.

The inducible degradation (ID) tag. (A) ID-tagged proteins are stable in the absence of HIV-2 protease but are degraded after proteolytic removal of the protecting peptide. (B) The ID tag is composed of two parts, a mutated mycobacterial SsrA sequence and a protecting peptide, separated by an HIV-2 protease recognition site. A c-myc epitope at the N terminus and a FLAG epitope at the C terminus of the ID tag permit immunodetection of target proteins. (C) Fluorescence of GFP fused to the ID tag in M. smegmatis carrying an inducible HIV-2 protease. Fluorescence is lost on overnight induction of HIV-2 protease using anhydrotetracycline (ATc). (D) The Zeocin resistance (Zeo) protein is efficiently degraded by the ID tag. M. smegmatis mc²155 containing both pGH1000A::zeo-ID and pMC1s::WT-HIV2Pr was grown in 96-well plates with varying concentrations of Zeocin or ATc. Expression of HIV-2 protease decreases the observed MIC (the concentration of antibiotic necessary to block conversion to a pink color).

Fig. 2.

Degradation of endogenous proteins fused to the ID tag. Western blot analysis of lysates carrying ID tag fusions to target proteins in the presence or absence of inducer (ATc) was performed using α-c-myc antibody to visualize ID-tagged proteins or α-ClpP antibody as a loading control. (A) Samples were collected 15 h after the addition of inducer. Loading of each lane was normalized using culture OD. (B) Semiquantitative Western blot of serially diluted lysates from strains grown in the absence of inducer compared with induced lysates (far right lane). (C) Time course of degradation. Lysates were collected at the indicated time points and normalized by culture OD.

Fig. 3.

Growth of strains after protein depletion. Strains carrying ID tags fused to GyrA, KasA, and RpoB were grown to midlog phase and preincubated with ATc for 12 h to allow protein degradation to proceed. Strains harboring ID tag fusions to Alr, DFHR, and InhA were not preincubated. To measure growth, strains were diluted 1:100 into fresh medium with or without ATc, and samples were removed at indicated time points for cfu determination. Insets show the amount of remaining protein at indicated time points as determined by Western blotting using anti–c-myc antiserum. A strain carrying the HIV-2 protease gene but no ID tag grew normally in the presence of ATc (Fig. S4).

Fig. 4.

MIC to antibiotics and enzymatic activities in protein-depleted strains. (A) Antibiotic susceptibilities of strains after protein depletion. White (WT), hatched (Alr), cross-hatched (DHFR), and black (GyrA) bars represent log₂ MIC values after depletion of designated proteins. (B) Strains carrying ID tag fusions to DHFR (Left) and Alr (Right) were assayed for enzymatic activity after overnight culture with and without targeted degradation. Because we used crude lysates, background for the DHFR assay was determined in the presence of saturating DHFR inhibitor, methotrexate (MTX; 1 μM). Background for the alanine racemase assay was determined by omitting the substrate, d-alanine.

Fig. 5.

Metabolites affected by DHFR depletion or trimethoprim treatment. (A) Biochemical pathways related to DHFR. (B) Profiles of 12 intracellular metabolites in M. smegmatics treated with trimethoprim or depleted of DHFR. Relative levels are expressed as the log ratio of signal intensity normalized to the control sample, which was untreated cells. For dUMP, AICAR, and homocysteine, the numbers are in log₁₀, and for the rest metabolites, the numbers are in log₂. Both drug treatment and DHFR depletion were performed on the same strain, which has an ID-tagged DHFR and inducible protease on the chromosome. Protease-only control cells were induced for protease but not depleted for DHFR. Trimethoprim cells (B Upper) were treated with various concentrations of trimethoprim but not depleted for DHFR. DHFR-KD + trimethoprim (B Lower) indicates bacteria grown in the presence of various concentrations of trimethoprim and ATc 50 ng/mL, which depletes DHFR to less than 2% of its physiological concentration. This experiment was performed in duplicate, and this result is representative of three independent experiments. Metabolite signal intensities were normalized to total protein in the lysates.