Environmental conditions which L. monocytogenes is exposed to prior to host invasion are decisive for its infective potential as virulence gene expression is generally down regulated after cold shock or in rich medium
[62, 63]. In contrast, exposure to high temperature (42°C), oxidants, entry into stationary phase induces the transcription of virulence genes, or oxygen limitation increases invasiveness 100-fold
. Stimuli for prfA induction include body temperature, low iron concentration, high osmolarity, activated charcoal, oxidative stress and conditions simulating the gastrointestinal system
[13, 14, 65–67], whereas fermentable sugars repress PrfA-dependent virulence genes
Our finding of prfA induction upon acid shock contrasts the outcome of previous studies
[15, 68] whose experimental conditions, however, are not comparable to those used in our work. Cells had been taken from stationary phase or from cultures grown to exponential phase in the presence of low pH, thus allowing the cells to adapt to acid. Likewise, Garner et al. reported an attenuated invasiveness of L. monocytogenes for Caco-2 cells, when the bacteria were grown at pH 5.5 in the presence of organic acids, and Rieu et al.
 demonstrated a decrease in virulence gene transcription after 5 h at pH 4.0 achieved with acetic acid. In both studies, listerial cells had been adapted to low pH previous to the experiment. These results are in line with our data showing prfA transcription levels and listerial invasiveness of epithelial cells to decrease to nearly pre-shock levels after adaptation to low pH (Figures
6). However, conducting a short time experiment, Werbrouck et al. failed to detect prfA induction after 1 h acid shock to pH 5.5. This conflicting finding might be explained by their use of organic acids that are much more harmful to the bacteria. To mimic gastric conditions, we, in contrast, added inorganic HCl to acid shock L. monocytogenes. The observed changes do not reflect growth-phase dependent physiological changes because listerial growth after acid shock was not observed for at least 1 hour (data not shown); therefore, the transcriptional response appears acid-shock specific. The prfA inducibility by HCl at 37°C as demonstrated here might be considered as an imitation of conditions during the stomach passage where acid is an early signal for a possible host entry, thus preparing bacteria for infection
We assume that in contrast to a temporary acid shock, prolonged mild acid conditions are often encountered by saprophytic bacteria in the environment, namely upon geological processes, fermentation of plant material, activity of plant roots or food processing
[7, 17, 72]. As a result of this acid adaptation process, virulence gene expression probably decreases in L. monocytogenes. Due to the ATR, the acid adapted listeriae also show an enhanced capacity to survive the gastric barrier as reflected by higher survival rates 15 min after intragastric infection of mice
. In this case, a further signal for virulence gene induction occurs after stomach exit, namely exposure to short chain fatty acids accompanied with pH neutralization
An unexpected result of this study was the induction of prfA and other in vivo-relevant genes at temperatures lower than 37°C after acid shock (Figures
4). Johansson et al. reported that prfA mRNA is stable at 30°C, but PrfA is only formed at 37°C
. According to Loh et al.
, PrfA expression is approximately 16-times lower at 30°C compared to 37°C and not detectable in L. monocytogenes cultivated at 20°C in BHI medium. However, using a prfA-promoter fusion to gfp, substantial expression of the reporter protein at 30°C in the heterologous system of E. coli was visible
. This suggests PrfA expression to be generally possible at temperatures below 37°C. Accordingly, it was argued that prfA upregulation occurs most likely at the σB-dependent P2 promoter and not via the σA-dependent P1 promoter, producing monocistronic prfA mRNA which is not blocked at temperatures below 30°C
A remarkable high number of genes involved in virulence, controlled by one of the main virulence regulators or induced during intracellular growth, also positively responded to acid shock at both temperatures, indicating that this signal contributes to the adaptation of L. monocytogenes during infection. A differential temperature response was observed for few genes only (Tables
2), and we hypothesize that this finding points to their requirement in specific hosts. While zinA, fbpA, clpC, sod and arpJ at 37 °C might specifically be induced in mammalian hosts, the low temperature-induced genes frvA, btlB, purB and glpK
may indicate a role in invertebrates. A set of up regulated genes identified here is influenced by σB. σB is not only responsible for listerial survival under acid stress conditions
, but for the transcription of several in vivo-relevant genes such as bsh, inlAB and opuCA[28, 35, 51, 77]. Remarkably, σB contributes to virulence and gastrointestinal spread, but not systemic infection
Uptake of L. monocytogenes into non-phagocytotic cells is mainly mediated by InlAB
[27, 28]. In agreement with the above reported increased invasiveness at 25°C, we observed a transient induction of inlA and inlB (e.g., at 60 min 6.5-fold for inlA and 10.9-fold for inlB; Figure
3), despite a normally lesser transcription at lower temperatures
[19, 79]. Enough InlAB seems to be produced for invasion after acid shock
. We detected a 12-fold increase in cell culture invasiveness one hour after acid shock at 25°C, compared to a modest 3.1-fold increase after acid shock at 37°C or at 30°C
[9, 80]. In contrast to acidic stress at 37°C
, listerial cells adapted to acid lost their higher potential to invade Caco-2 cells. σB, which is active at low environmental pH, contributes to invasion by controlling inlAB. This is in accordance with our finding that σB-dependent genes are induced upon acid shock.
The above described data prompted us to investigate acid shock induced prfA transcription at even lower temperatures of 18°C and 10°C. Unexpectedly, lower temperatures corresponded with higher folds of prfA induction when compared to unshocked cells at the same temperature (Figure
1). A similar response was observed for six other L. monocytogenes strains. We wondered whether the increase of virulence factor transcription due to acidic conditions at temperatures below 37°C confers increased pathogenicity of L. monocytogenes under yet unknown conditions. To answer this question, we applied a nematode killing assay. When we fed acid-shocked L. monocytogenes to C. elegans we could observe that the nematodes died faster compared to nematodes fed with unshocked bacteria. These observations clearly showed the biological relevance of virulence gene induction after acid shock at room temperature. D. melanogaster and its cells grown at 30°C have also successfully been introduced as a model for listerial infection
[18, 20]. Therefore, it could be hypothesized that other poikilothermal animals are hosts of L. monocytogenes, although virulence genes appear generally down regulated at low temperature
Indeed, Listeria spp. have been isolated from reptiles, amphibians, fish, as well as snails, crustaceans, diptera and leeches in the past
[82–85], but to our knowledge no acute illness of such hosts upon listerial infection has been reported. Interestingly, the above mentioned poikilothermal animals acidify their stomach or gut content during digestion
[86–91], and the resulting low pH may serve as a signal of host entry and virulence gene induction at environmental temperatures.