Figure 1.
The standard model for the action of the GAD system.
(a) A membrane bound antiporter carries glutamate into the cell in exchange for GABA. A cytosolic decarboxylase enzyme converts glutamate to GABA, with a consumption of H+. (b) The genomic structure of the genes encoding the GAD system in L. monocytogenes EGD-e.
Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study.
Figure 2.
Acid survival of L. monocytogenes gad mutants.
Stationary phase EGDm (a) and 10403S (b) Δgad mutants were challenged at pH 2.5. Cell counts were taken every 20 min. Values are the means of data from three individual cultures, with the cell counts for each culture being the means of counts from three platings. Relative transcript levels of EGDm (c) or 10403S (d) gadD1 (dark grey fill), gadD2 (hatched) and gadD3 (grey) genes to 16S gene prior to acid exposure in each mutant strain. Error bars represent the standard error from the mean value of three individual biological repeats. The numbers over the bar charts (c & d) indicate the p-value for the difference between each gene expression compared to wild-type levels as determined by student’s t-test.
Figure 3.
GABA production from L. monocytogenes gad mutants.
(a) Production of GABAe by EGDm and 10403S gad mutants with (grey) or without (black) 1 h exposure to acid at pH 4.0 (EGDm) or pH 3.5 (10403S). (B) Production of GABAi by EGDm and 10403S gadD mutants with (grey) or without (black) 1 h exposure to acid at pH 4.0 (EGDm) or pH 3.5 (10403S). Dashed horizontal lines indicate the detection limits for GABA in each experiment. Error bars represent the standard deviation from the mean of three individual biological repeats for each sample. An asterix represents signifcant difference of less than 0.05 between a given mutant and respective wild-type as determined by a student’s t-test.
Figure 4.
Relative expression of gad genes in response to acid treatment.
Expression of gadD1, gadD2, gadD3 and lmo2230 relative to expression of the 16S rRNA gene prior to, 15 min and 30 min after exposure in BHI broth to pH 4.0 (EGDm (a)) or pH 3.5 (10403S (b)). Error bars represent the standard error in the mean of 3 independent biological repeats. Differences found to be significant between the genes at any time-point for each strain are shown with * Significance was determined where p <0.05 as determined by a Student’s t-test.
Figure 5.
Acid survival and GABAi production of L. monocytogenes EGDm double GAD system mutants indicates a key role for gadD3.
(a) Stationary phase EGDm gadD mutants were acidified to pH 2.5 with 3 M HCl in BHI broth. Cell counts were taken every 20 min. Values are the means of data from three individual cultures, with the cell counts for each culture being the means of counts from three platings. Error bars represent the standard deviation from the mean value for each time-point. (b) Stationary phase EGDm gad mutants were acidified (grey) or not acidified (black) with 3 M HCl to pH 4.0 and GABAi accumulation was quantified. Error bars represent the standard deviation from the mean of three independent biological replicates.
Figure 6.
Infection of Balb/C mice with EGDm GAD system mutants.
Plate counts of surviving EGDm GAD system mutants 3 days post infection from female Balb/C mice (n = 5). Isolated from the liver, spleen, mesenteric lymph node (MLN) and faeces. Significant differences (*) between wild-type and mutants were determined using one-way ANOVA.
Figure 7.
Growth of GAD system mutants in THP-1 macrophages.
Growth of EGDm (a) and 10403S (b) GAD system mutants inside THP-1 macrophages over 7 h. Counts are recorded 2 h post co-incubation of THP-1 with bacteria at an MOI of 10 (106 bacteria; black arrow). Error bars represent the standard deviation from the mean of at least 4 biological replicates for each strain and time-point. Significant differences (*; p <0.05) were determined using one-way ANOVA.