Pyrazinamide Susceptibility Is Driven by Activation of the SigE-Dependent Cell Envelope Stress Response in Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We found that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, exposure to peptidoglycan synthesis inhibitors, such as beta-lactams and d-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response. IMPORTANCE For decades, pyrazinamide has served as a cornerstone of tuberculosis therapy. Unlike any other antitubercular drug, pyrazinamide requires an acidic environment to exert its action. Despite its importance, the driver of this conditional susceptibility has remained unknown. In this study, a genome-wide approach revealed that pyrazinamide action is governed by the cell envelope stress response. This observation was validated by orthologous approaches that demonstrate that a central player of this response, SigE, is both necessary and sufficient for potentiation of pyrazinamide action. Moreover, constitutive activation of this response through deletion of the anti-sigma factor gene rseA or exposure of bacilli to drugs that target the cell wall was found to potently drive pyrazinamide susceptibility independent of environmental pH. These findings force a paradigm shift in our understanding of pyrazinamide action and open new avenues for improving diagnostic and therapeutic tools for tuberculosis.

Keywords: cell envelope; drug discovery; drug resistance mechanisms; genomics; metabolism; tuberculosis.

FIG 1

Genes associated with M. tuberculosis PZA susceptibility by Tn-seq. Libraries of 2 × 10⁵ independent M. tuberculosis H37Rv himar1 insertion mutants (4-fold saturation) were plated on 7H9 agar without (a) or with (b) POA. Genomic DNA was extracted, processed, and sequenced as described by Minato et al. (37). (a and b) Mean abundance relative to total TA insertion read counts from two independent replicates. (c) Log₂ fold changes in mean relative abundance from panel b compared to panel a by gene and respective −log₁₀ P value (dotted line at P = 0.05). (d to i) Read count comparisons for transposon insertions in panD (d), P_clpC1 (e), B11 (f), crp (g), sigE (h), and rpoC (i).

FIG 2

Deletion of rseA in M. tuberculosis confers constitutive susceptibility to PZA. M. tuberculosis H37Rv (wild type, black circles) and the ΔrseA strain (blue squares) were grown in 7H9 at pH 5.8 (a and b) or pH 6.6 (c to e) and exposed to PZA (a and c) or POA (b and d) for 14 days or INH for 7 days (e). OD₆₀₀ was measured, and relative growth was determined by dividing by the value of the no-drug control. Data are means and standard deviations (SD) for 3 biological replicates. *, P < 0.05, and **, P < 0.0002, by 2-tailed Student's t test.

FIG 3

Distribution of transposon insertions and natural sequence polymorphisms observed in clinical isolates in SigE functional domains. Location of Tn-seq insertions enriched under POA pressure and nonsynonymous single nucleotide polymorphisms (SNPs) observed across PZA-monoresistant clinical isolates with respect to SigE domain architecture imported from InterPro and curated from recent literature (51). Locations of all missense mutations present in at least one isolate and all TA insertion sites with greater relative abundance in POA-containing media relative to the 7H9 control are depicted.

FIG 4

Peptidoglycan synthesis inhibitors potentiate antitubercular activity of pyrazinamide. M. tuberculosis H37Rv (a and d to f), Erdman (b) and H37Rv ΔsigE (c) were grown in 7H9 at pH 6.6 with various concentrations of PZA and either meropenem/clavulanate (a to c), d-cycloserine (d), ethambutol (e), or isoniazid (f) in checkerboard format. OD₆₀₀ was determined after 7 days of incubation. Plots were generated based on the average fractional inhibitory concentration (FIC) calculated from two biological replicates. The lowest FIC index (FICI) values are indicated in red. Line of additivity is shown in gray (FICI of 1).

FIG 5

Model for low pH-mediated potentiation of PZA susceptibility. (a) Under conditions of balanced growth, M. tuberculosis is in a nonstressed state, resulting in PZA resilience. (b) Under conditions of low pH or other cell wall stress-inducing conditions, signaling through PknB mediates phosphorylation of RseA, which is subsequently degraded by ClpC1P1P2, liberating SigE. Expression of the SigE regulon results in metabolic remodeling that poises bacilli in a PZA-susceptible state. (c) Failure to activate the SigE response prevents cell wall stress-driven metabolic remodeling, resulting in PZA resistance.