Phosphorylation on PstP Regulates Cell Wall Metabolism and Antibiotic Tolerance in Mycobacterium smegmatis

Abstract

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the serine/threonine phosphatase PstP, in the model organism Mycobacterium smegmatis We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phosphomimetic mutation, pstP T171E, slows growth, misregulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.IMPORTANCE Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

Keywords: CwlM; Mycobacteria; PstP; antibiotic tolerance; cell wall metabolism; dephosphorylation; mycolic acid metabolism; peptidoglycan metabolism; serine/threonine phosphatase; starvation.

FIG 1

Phosphosite T171 on PstP affects growth. (A) Schematic of the crystal structure of PstP from M. tuberculosis (PstP_Mtb) (55) (PDB code 1TXO). The threonine (T) sites on PstP_Mtb phosphorylated by the kinases PknA and PknB (56) are highlighted on the structure as follows: red, PstP_Mtb T137 (T134 in PstP_Msmeg); blue, PstP_Mtb T141 (T138 in PstP_Msmeg); and green, PstP_Mtb T174 (T171 in PstP_Msmeg). (B) Doubling times of strains containing pstP_Msmeg WT, phosphoablative mutant alleles pstP_Msmeg T134A, T138A, and T171A, and phosphomimetic mutant alleles pstP_Msmeg T134E, T138E, and T171E. Each dot is the mean of doubling times from two or three different experiments on different dates of a single isolated clone. The error bars represent the standard deviation. ***, P = 0.0003.

FIG 2

PstP_Mtb dephosphorylates CwlM_Mtb. (A) Antiphosphothreonine (anti-P-Thr) and anti-His Western blots of in vitro phosphatase reactions with His-PstP_cWT_Mtb (top panel), His-PstP_cT174E_Mtb (middle panel), and no-phosphatase control (bottom panel) against phosphorylated His-SUMO-CwlM_Mtb. The assay was performed at least twice with two individually purified batches of each phosphatase; one set of images is shown here. (B) Quantification of relative intensities of anti-P-Thr over anti-His on Western blots. P values were calculated using two-tailed, unpaired t test. All the P values of WT versus T171E at any given time were nonsignificant. P values of WT versus T171E at 5 min = 0.683; 10 min = 0.809; 23 min = 0.934; 40 min = 0.831; 60 min = 0.876; and 90 min = 0.545. The error bars represent standard error of means.

FIG 3

Phosphosite T171 on PstP_Msmeg is important in regulating cell length. (A) Quantification of cell lengths of isogenic pstP allele strains (T17A, T171E, and WT) grown in 7H9 medium in log phase. One hundred cells from each of three biological replicates of each pstP allelic variant were measured. P values were calculated by unpaired t test; ****, P = 0.000005. (B) Representative phase images of cells from panel A. (C) Quantification of cell lengths of isogenic pstP allele strains (T17A, T171E, and WT) after starvation in HdB with no glycerol for 5.5 h. One hundred cells from each of three biological replicates of each pstP allelic genotype were measured. P values were calculated by unpaired t test; ****, P = 0.000003. (D) Representative phase images of cells from panel C.

FIG 4

Phosphosite T171 of PstP alters cell wall staining. (A and B) Quantification of mean intensities of HADA (A) and DMN-Tre (B) signals of pstP allele strains (WT, T17A, and T171E) in log-phase cells, with representative cells below. In panel B, P values of both WT versus T171A and WT versus T171E were 0.000001. (C and D) Quantification of mean intensities of HADA (C) and DMN-Tre (D) signals of starved pstP allele strains (WT, T17A, and T171E) after 5.5 h in HdB with no glycerol, with representative cells below. In panel C, P values of WT versus T171A = 0.0002 and WT versus T171E = 0.000001. (E and F) Intensity profiles of HADA (E) and DMN-Tre (F) signals in cells from pstP allele strains (WT, T17A, and T171E) after starving for 5.5 h in HdB with no glycerol. The shaded regions denote standard deviations and the solid lines represent the mean intensity values. Signal intensities from at least 100 cells from each of three biological replicates of every pstP allelic variant genotype were analyzed in MicrobeJ. In panels A to D, the values of the intensities are represented in percentages of the maximum value of all intensities for all strains either in log phase or during starvation. P values were calculated by a two-tailed, unpaired t test on all 300 values of each pstP allelic variant genotype (WT, T17A, and T171E) using GraphPad Prism (v7.0d).

FIG 5

Phosphosite T171 of PstP plays a role in antibiotic sensitivity. Survival of pstP allele strains (WT, T17A, and T171E) in different media and antibiotics. (A) Log-phase culture in 7H9 medium treated with 8 μg/ml of meropenem. P values of WT versus T171E at 3 h = 0.043; WT versus T171A at 48.5 h = 0.001; and WT versus T171E at 48.5 h = 0.018. (B) Starvation culture in HdB medium (no glycerol, 0.05% Tween) for 5.5 h and then treated with 45 μg/ml of meropenem. P values of WT versus T171E at 16 h = 0.003; 23 h = 0.007; 26.3 h = 0.004; and 41 h = 0.046. (C) Log-phase culture in 7H9 medium treated with 100 μg/ml of d-cycloserine. P values of WT versus T171E at 8 h = 0.032. (D) Starvation culture in HdB medium (no glycerol, 0.05% Tween) for 5.5 h and then treated with 900 μg/ml of d-cycloserine. P values of WT versus T171E at 3.5 h = 0.046; 14 h = 0.001; and 18 h = 0.022. (E) Log-phase culture in 7H9 medium treated with 10 μg/ml of isoniazid. P values of WT versus T171E at 4 h = 0.008; WT versus T171E at 29 h = 0.002; and WT versus T171A at 35 h = 0.052. (F) Starvation culture in HdB medium (no glycerol, 0.05% Tween) for 5.5 h and then treated with 90 μg/ml of isoniazid. P values of WT versus T171A at 15.3 h = 0.009. (G) Log-phase culture in 7H9 medium treated with 50 μg/ml of trimethoprim. P values of WT versus T171E at 4 h = 0.023; 8 h = 0.013; 12 h = 0.005; 22.5 h = 0.015; 26 h = 0.005; 30 h = 0.017; and 34 h = 0.002. (H) Starvation culture in HdB medium (no glycerol, 0.05% Tween) for 5.5 h and then treated with 360 μg/ml of trimethoprim. P values of WT versus T171A at 18 h = 0.023. All experiments were done with three biological replicate strains of each pstP allelic variant (T171A, T171E, and WT) at least twice. One representative trial (performed with three biological replicate strains of each pstP allelic variant genotype) is shown. All P values were calculated using two-tailed, unpaired t test. All error bars represent standard deviation (SD).