Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans

Abstract

Background: The saprophytic pathogen Listeria monocytogenes has to cope with a variety of acidic habitats during its life cycle. The impact of low-temperature coupled with pH decrease for global gene expression and subsequent virulence properties, however, has not been elucidated.

Results: qRT-PCR revealed for the first time a transient, acid triggered prfA induction of approximately 4-fold, 5.7-fold, 7-fold and 9.3-fold 60 to 90 min after acid shock of L. monocytogenes at 37°C, 25°C, 18°C, and 10°C, respectively. Comparable data were obtained for seven different L. monocytogenes strains, demonstrating that prfA induction under these conditions is a general response of L. monocytogenes. Transcriptome analysis revealed that the in vivo-relevant genes bsh, clpP, glpD, hfq, inlA, inlB, inlE, lisR, and lplA1 as well as many other genes with a putative role during infection are transiently induced upon acid shock conducted at 25°C and 37°C. Twenty-five genes repressed upon acid shock are known to be down regulated during intracellular growth or by virulence regulators. These data were confirmed by qRT-PCR of twelve differentially regulated genes and by the identification of acid shock-induced genes influenced by σB. To test if up regulation of virulence genes at temperatures below 37°C correlates with pathogenicity, the capacity of L. monocytogenes to invade epithelial cells after acid shock at 25°C was measured. A 12-fold increased number of intracellular bacteria was observed (acid shock, t = 60 min) that was reduced after adaptation to the level of the unshocked control. This increased invasiveness was shown to be in line with the induction of inlAB. Using a nematode infection assay, we demonstrated that Caenorhabditis elegans fed with acid-shocked L. monocytogenes exhibits a shorter time to death of 50% (TD50) of the worms (6.4 days) compared to infection with unshocked bacteria (TD50 = 10.2 days).

Conclusions: PrfA and other listerial virulence genes are induced by an inorganic acid in a temperature-dependent manner. The data presented here suggest that low pH serves as a trigger for listerial pathogenicity at environmental temperatures.

Figure 1

Transcriptional induction of prfA upon acid shock at different time points. Samples were taken before (t = 0) and at five time points until two hours after acid shock. Mean values of fold expression ± standard deviation calculated as ratio between prfA transcription after and before HCl treatment are presented. To obtain a sample of adapted cells, acid-shocked L. monocytogenes (120 min pH 5) were diluted and grown at pH 5.2 to OD_600nm = 0.5. Expression of prfA at 10°C and 18°C was measured by real time qRT-PCR in replicates of three and at 25°C and 37°C in two biologically independent experiments, respectively. The prfA induction was not determined at some time points (e.g., for 10 and 18°C at 15 min, 25 and 37°C at 90 min, and 10 and 18°C for the adaptation sample).

Figure 2

prfA induction levels in L. monocytogenes strains. The transcriptional change of the prfA mRNA from five additional L. monocytogenes strains belonging to serotypes 1/2a, 3a and 4b at 18°C and 60 min after acid shock to pH 5.0 was measured by qRT-PCR in three independent experiments. The value for strain EGDe from Figure 1 is given in the last column as reference.

Figure 3

Up regulation of internalin and other in vivo-relevant genes upon acid shock. Transcriptional induction of selected genes transcriptionally induced after acid shock at 25°C (black columns) compared to acid shock at 37°C (grey columns) as deduced by microarray analysis. All genes have experimentally been demonstrated to play a role in listerial virulence (Additional file 2). Samples were taken 15, 30, 60 and 120 min after acid shock, and from acid-adapted cells. Two biological and two technical replicates were performed on microarray for each time point measured.

Figure 4

Heat map of genes influenced by PrfA or σ^B, or of intracellularly induced genes. Average expression of in vivo-relevant expression after acid shock at 25°C and at 37°C. In both experimental settings, samples were taken 15, 30, 60 and 120 min after acid shock, and from acid-adapted cells. Genes have been grouped according to their putative regulation by PrfA and σ^B, as well as to up regulation during intracellular growth. Up regulation is colored in shades of red, down regulation in shades of blue. “No change” is indicated by black and missing values by grey coloring.

Figure 5

Correlation of microarray data and qRT-PCR analysis of selected genes. The following genes were selected for microarray validation by qRT-PCR: prfA, hly, flhB, cheA, lmo0109, lmo0847, lmo1740, rsbR, lmo1389, lmo1997, lmo2434, and lmo2784 (see Additional file 3 for more functional details). The validated genes checked at various time points (♦) displayed a significant correlation (level of correlation = 0.966) between the data obtained by microarray analysis and by qRT-PCR. Three independent qRT-PCR experiments were performed.

Figure 6

Invasiveness of L. monocytogenes after acid shock at 25°C. Invasion assays were performed with Caco-2 cells. Untreated and acid-shocked EGDe cells were used for infection at an MOI of 10. The invasion process was blocked after 1 h by cell washing and gentamycin treatment, and the epithelial cells were lysed to enumerate intracellular listeriae. The percentage of colony forming units (cfu) derived from acid treated inoculate as normalized to the control using untreated cells. Standard deviations of three independent invasion assays, performed at least as triplicates, are shown. In each experiment, the significance value was below 0.05 (p < 0.05) as calculated by the Student’s t-test. The fold difference in invasion is approximately 6-fold at 30 min and 12-fold at 60 min after acid shock.

Figure 7

Nematode infection assays. Death curves of nematodes fed with acid-shocked L. monocytogenes (◊) and unshocked control cells (■). Surviving worms were transferred daily to freshly prepared acid-shocked or unshocked L. monocytogenes for up to 14 d. The error bars are from four independent experiments using 20 worms in each condition, totaling 160 worms.