Analysis of murinized H7858 <i>L. monocytogenes</i>.

<p>(A) The murinized H7858 strain has a greater ability to infect the mouse by the oral route compared to the wild-type strain. BALB/c mice were orally infected with 1 x 10¹⁰ CFU with either the murinized and wild-type H7858 strain. Bacterial CFU in the liver (black bars) and spleen (grey bars) were enumerated at 3 days post-infection. N=5 mice per group and the values are the mean and standard deviation. (B) Invasion assay of Caco₂ cell line by wild-type and murinized H7858. Under our conditions tested the murinized strain had a decreased ability to invade the Caco₂ cell line. This was carried out in triplicate and the values are the mean and standard deviation. * indicates P<0.05 relative to control strain.</p

Insertion sites of transposon mutants identified in the GI STM screen.

<p>The diagram was drawn approximately to scale using <i>Listeria monocytogenes</i> H7858 genome sequence data (TIGR). Open reading frames (shaded in grey) are genes with transposon insertion. Black arrowheads represent the approximate location of transposon insertion. White open reading frames are flanking genes. Lollipops indicate predicted terminator locations. The number correspond to the lmOh7858 annotated numbers in the H7858 genome.</p

Overview of the STM system.

<p>(A) A unique STM tag was created with Xho1 restriction enzyme sites and integrated into the mariner plasmid pJZ037. In total there were 48 unique tags created in an <i>E. coli</i> background and then transformed into the <i>L. monocytogenes</i> H7858m strain. (B) The mutants were pooled and screened in BALB/c mice where the liver, spleen and mesenteric lymph nodes were removed at 1 day post-infection. The IP and OP pools were analysed by PCR to identify non-colonising mutants.</p

In vivo analyses of individual Tn mutants after oral infection.

<p>The kinetics of infection was analyzed on day 1 (A) (C) and day 3 (B) (D) post infection. Bacterial infection was monitored in the liver, spleen and mesenteric lymph nodes. Values are the mean and standard deviation of 5 mice and CFU per organ. ND, not detected. * indicates P<0.05 relative to wild-type control.</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

Primers used in this study.

<p>Primers used in this study.</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

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

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