Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in listeria monocytogenes egd-e and 10403s: roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for c-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GAD(i)), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GAD(i)/GAD(e)). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GAD(i) but not GAD(e) the results indicate that the GAD(i) system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains

The standard model for the action of the GAD system.

<p>(<b>a</b>) 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>b</b>) The genomic structure of the genes encoding the GAD system in <i>L. monocytogenes</i> EGD-e.</p

Acid survival and GABA_i production of <i>L. monocytogenes</i> EGDm double GAD system mutants indicates a key role for <i>gadD3</i>.

<p>(<b>a</b>) Stationary phase EGDm <i>gadD</i> 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>b</b>) Stationary phase EGDm gad mutants were acidified (grey) or not acidified (black) with 3 M HCl to pH 4.0 and GABA_i accumulation was quantified. Error bars represent the standard deviation from the mean of three independent biological replicates.</p

Infection of Balb/C mice with EGDm GAD system mutants.

<p>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.</p

Growth of GAD system mutants in THP-1 macrophages.

<p>Growth of EGDm (<b>a</b>) and 10403S (<b>b</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 (*; <i>p</i> <0.05) were determined using one-way ANOVA.</p

GABA production from <i>L. monocytogenes gad</i> mutants.

<p>(<b>a</b>) Production of GABA_e 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>B</b>) Production of GABA_i by EGDm and 10403S <i>gadD</i> 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 <i>t-</i>test.</p

Acid survival of <i>L. monocytogenes gad</i> mutants.

<p>Stationary phase EGDm (<b>a</b>) and 10403S (<b>b</b>) Δ<i>gad</i> 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 <b>(c)</b> or 10403S (<b>d</b>) <i>gadD1</i> (dark grey fill), <i>gadD2</i> (hatched) and <i>gadD3</i> (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 (<b>c & d</b>) indicate the <i>p</i>-value for the difference between each gene expression compared to wild-type levels as determined by student’s <i>t</i>-test.</p

Relative expression of <i>gad</i> genes in response to acid treatment.

<p>Expression of <i>gadD1</i>, <i>gadD2</i>, <i>gadD3</i> and <i>lmo2230</i> 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 <b>(a)</b>) or pH 3.5 (10403S <b>(b)</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 <i>t</i>-test.</p

Primers used in this study.

<p>Primers used in this study.</p