Fig 1.
Methylglyoxal production and detoxification pathways.
(A) MG is formed mainly as a byproduct of glycolysis. In most organisms it is transformed from dihydroxyacetone phosphate (DHAP) by methylglyoxal synthase (MGS). (B) MG production can also result from the metabolism of lipids and proteins. (C) MG detoxification occurs mainly through the glyoxalase system that consists of two enzymes glyoxalase A (GloA) and glyoxalase B (GloB). MG can react non-enzymatically with reduced glutathione (GSH) to form a hemithioacetal which is transformed to S-lactoylglutathione by GloA. S-lactoylglutathione activates potassium efflux pumps that acidify the cytoplasm and confer protection against MG. GloB then transforms S-lactoylglutathione to D-lactate, recycling GSH in the process. Adapted from Allaman et al, 2015 [7].
Table 1.
Genes identified in small-plaque screen.
Fig 2.
In vitro phenotypes of glyoxalase mutants.
(A) Plaque area measured 3 days post-infection as a percentage of wild-type. Mean and standard error of the mean (SEM) pooled from three independent experiments is shown (n = 30). (B) Broth growth curve of indicated L. monocytogenes strains grown in BHI medium at 37°C with shaking. Mean and standard deviation of three independent experiments is shown. BMM’s were infected at an MOI of 0.25 with L. monocytogenes without treatment (C) or treated with PAM3CSK4 (D) and intracellular CFU were enumerated at different time points. Data are mean and SEM of three technical replicates of three independent experiments. For both panels, statistical significance is shown for ΔgloA compared to wild-type L. monocytogenes. (E) Sensitivity to MG (20% v/v) as measured by growth inhibition in a disk diffusion assay as percentage of wild-type. Data are mean and SEM of at least three independent experiments. For all experiments p values were calculated comparing to the wild-type bacteria using an unpaired Student’s t- test; *P < 0.05, **P < 0.01, ***P < 0.001, **** indicates P <0.0001.
Fig 3.
Virulence of glyoxalase mutants in vivo.
(A and B) Female CD-1 mice were infected with 105 CFU L. monocytogenes. Spleens (A) and livers (B) were harvested 48 hours post-infection and CFU were counted. Data and median represent three pooled experiments (n = 15). Data area mean and SEM of at least three independent experiments. For all experiments p values were calculated comparing to the wild-type bacteria using an unpaired Student’s t- test; *P < 0.05, **P < 0.01, ***P < 0.001, **** indicates P <0.0001.
Fig 4.
Mutation rates in L. monocytogenes glyoxalase mutants.
Rifampicin mutation frequencies were determined in response to MG exposure and sodium benzoate. L. monocytogenes were grown overnight in BHI broth (A) or defined medium (cLSM) (B) and plated on BHI agar containing 5ug/mL of rifampicin. Mutation frequency was calculated as the ratio between the number CFU enumerated on the rifampicin plates and the total number of bacteria plated. Mean and SEM of two technical replicates from three independent experiments are shown. For all experiments p values were calculated comparing to the untreated bacteria using an unpaired Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, **** indicates P <0.0001.
Fig 5.
Glutathione, gshF mRNA and actA mRNA levels in glyoxalase mutants.
(A) L. monocytogenes strains were grown to mid-log in defined media and challenged with 0.4 or 1.2 mM MG. Intracellular glutathione concentration was measured relative to wild-type at different time points post-challenge. Mean and SEM are shown of two technical replicates from two independent experiments. (B-C) Gene expression determined 15 minutes after MG exposure measured by RT-PCR. (D) Gene expression determined during BMMs infection measured by RT-PCR. Data are mean and SEM of two technical replicates from two independent experiments. For all experiments p values were calculated comparing to the wild-type bacteria using an unpaired Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, **** indicates P <0.0001.