The critical role of S-lactoylglutathione formation during methylglyoxal detoxification in Escherichia coli

Abstract

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.

Fig. 1

Detoxification of MG in enteric bacteria. Schematic representation of the major pathways of MG synthesis and detoxification, including the link to KefGB. d-Lac, d-Lactate; P_i, inorganic phosphate.

Fig. 2

Mutant strain lacking GlxII exhibits a reduced capacity to detoxify MG, but is not more sensitive to the electrophile. A. Genomic context of the gloB gene in E. coli. The gloB gene (756 bp) encodes for the GlxII enzyme (EC 3.1.2.6, hydroxyacylglutathione hydrolase). The flanking genes mltD and yafS are transcribed divergently from gloB. Arrows indicate gene boundaries and transcriptional orientation. Genome co-ordinates are shown above the arrows. Bar and dashed lines indicate the deleted genomic region in the ΔgloB strain (MJF595). Arrows on the scale of the genome co-ordinates indicate promoter elements (−35 elements) for mltD and yafS as predicted by BPROM (see Supporting information). B. The gloB null mutant has impaired MG detoxification. Rate of MG detoxification does not change when GlxII is overexpressed. Cells from the parent (MJF274, ◊), ΔgloB (MJF595, ◆) and pGlxII () were grown to OD₆₅₀ of ∼0.4 in K_0.2 minimal media and diluted 10-fold into fresh media containing 0.7 mM MG. At intervals the medium was assayed for the disappearance of MG. The data are representative of three independent replicates. C. The gloB null mutant exhibits similar death kinetics to the parent strain upon MG stress. Cells overexpressing GlxII are more sensitive to MG. Cells from the parent (MJF274, ◊), ΔgloB (MJF595, ◆) and pGlxII () were grown exactly as in B and diluted into media containing 0.7 mM MG. Cell samples were taken at intervals and the number of viable cells determined. Data represent the mean of three independent replicates (standard deviations are shown).

Fig. 3

K⁺ efflux systems are hyperactive in a gloB null mutant. A and B. K⁺ efflux from the parent (MJF274, A) and ΔgloB (MJF595, B) upon exposure to different MG concentrations. Cells were grown to an OD₆₅₀ of ∼0.8 in K⁺-rich minimal medium (K₁₁₅), harvested and suspended in K⁺-free buffer. K⁺ efflux was measured in the absence (control; ▵, ▴) and in the presence of 0.7 mM (○, •) and 3 mM MG (◊, ◆). MG was added 2 min (indicated by arrow) after resuspension of cells in K⁺-free buffer. Control data were averaged for illustration. Data shown are representative of three independent replicates. At time zero MJF274 contained ∼494 ± 8 µmol K⁺ and MJF595 contained 485 ± 18 µmol K⁺ per gram dry cell weight. C. First order rate constants (k) for K⁺ efflux over a range of MG concentrations. K⁺ efflux from the parent (◊) and ΔgloB (◆) was measured using different MG concentrations (0.025–3 mM). Rate constants were determined over a period of 3 min after the addition of MG (t₂ to t₅; see also Experimental procedures) and multiplied by −1 for illustration purposes. Data represent the mean of three independent replicates (standard deviations are shown). Datasets for both strains were fitted using an exponential association function in the Origin 8.0 software {Equation: y = y₀ + A₁ × [1 − exp(−x/t₁)] + A₂ × [1 − exp(−x/t₂)]} and the output for each dataset is shown as a dashed line. D. First order rate constants (k) for K⁺ efflux of the parent, ΔgloB and pGlxII when treated with 0.7 mM MG. Rate constants were determined as for Fig. 3C. *Not significantly different to the rate of spontaneous K⁺ loss from cells untreated with MG.

Fig. 4

SLG accumulates rapidly to high levels in a gloB null mutant. A. Changes in GSH and SLG levels upon MG exposure were quantified in both the parent strain and the gloB null mutant. Cells were grown in K_0.2 minimal medium to an OD₆₅₀ of ∼0.8, handled as in K⁺ efflux assays and cells sampled at various time points. MG (0.2 mM) was added immediately after suspending cells in K₀ buffer (t_{0 s}). GSH (open symbols) and SLG levels (closed symbols) from the parent (○, •) and ΔgloB (MJF595, ◊, ◆) were quantified by LC-MS/MS. Data are representative of three independent replicates. Figures show metabolite concentrations as quantified in the extraction volume (see Experimental procedures); a concentration of 100 µM in the extraction volume equates to an intracellular concentration of ∼6.35 mM (see Experimental procedures). B–D. SLG levels in the parent (B), ΔgloB (C) and pGlxI (D) strains upon exposure to a range of MG concentrations. Cells were sampled 10 s (○, •), 2.5 min (□, ) and 5 min (◊, ◆) after addition of MG, and the metabolite pools quantified by LC-MS/MS. The mean and standard deviation of three independent replicate experiments is shown.

Fig. 5

Complex relationships exist between SLG levels, activation of K⁺ efflux systems and modulation of pH_i. A. First order rate constants for K⁺ efflux (k) from the ΔgloB mutant, and the parent were plotted against mean SLG levels at t_{10 s} after addition of selected MG concentrations. The data point in gray illustrates the lack of K⁺ efflux in the absence of SLG. B. The change in intracellular pH (ΔpH_i) in the parent (◊) and ΔgloB (◆) as a function of the concentration of exogenously applied MG. The ΔpH_i was calculated as (t_{10–14 min}) − (t_{15–18 min}) where MG was added at t_{15 min} for each MG concentration. The data shown are means ± s.e.m. Datasets for both strains were fitted using an exponential association function in the Origin 8.0 software [Equation: y = A₁ × exp(−x/t₁) + y₀] and the output for each dataset is shown as a dashed line. The adjusted R² values for the parent and ΔgloB were 0.76 and 0.96 respectively. C. First order rate constants (k) for survival of the parent (◊), ΔgloB (◆), pGlxII () and ΔgloB; ΔkefC; ΔkefB (MJF596, ▴) upon MG exposure were derived from viable cell counts over the first 60 min after addition of 0.7 mM MG. These data were plotted against the ΔpH_i calculated as described in B. Data plotted are means ± s.e.m. Data points were fitted using an exponential association function in the Origin 8.0 software [Equation: y = A₁ × exp(−x/t₁) + y₀]. The output is shown as a dashed line and the adjusted R² = 0.89. *As a consequence of the method used to determine pH_i and subsequently derive ΔpH_i, the absence of a drop in pH_i upon addition of MG, as is the case for MJF596, can lead to a negative value for ΔpH_i as the steady state pH_i measured over the time-course fluctuates around pH 7.8.

Fig. 6

Survival of gloB null mutant upon MG stress depends on the activity of K⁺ efflux systems. Cells that lack the K⁺ efflux systems KefGB and KefFC, in addition to GlxII, are highly sensitive to MG exposure. Cells from the parent (◊), ΔgloB (◆), MJF276 (kefB, kefC::Tn10; ) and MJF596 (ΔgloB, kefB, kefC::Tn10; ▴) were grown in K_0.2 minimal media, exposed to 0.7 mM MG and viable cells enumerated exactly as for experiments presented in Fig. 2. The mean and standard deviation of three independent experiments is shown.