The gram-positive bacterium Listeria monocytogenes is a food-borne pathogen of both public health and food safety significance. It possesses three small, highly homologous protein members of the cold shock protein (Csp) family. We used gene expression analysis and a set of mutants with single, double, and triple deletions of the csp genes to evaluate the roles of CspA, CspB, and CspD in the cold and osmotic (NaCl) stress adaptation responses of L. monocytogenes. All three Csps are dispensable for growth at optimal temperature (37°C). These proteins are, however, required for efficient cold and osmotic stress tolerance of this bacterium. The hierarchies of their functional importance differ, depending on the environmental stress conditions: CspA>CspD>CspB in response to cold stress versus CspD>CspA/CspB in response to NaCl salt osmotic stress. The fact that Csps are promoting L. monocytogenes adaptation against both cold and NaCl stress has significant implications in view of practical food microbial control measures. The combined or sequential exposure of L. monocytogenes cells to these two stresses in food environments might inadvertently induce cross-protection responses.

The cold shock protein (Csp) family comprises small, highly conserved proteins that bind nucleic acids to modulate various bacterial gene expressions. In addition to cold adaptation functions, this group of proteins is thought to facilitate various cellular processes to promote normal growth and stress adaptation responses. Three proteins making up the Listeria monocytogenes Csp family (CspA, CspB, and CspD) promote both cold and osmotic stress adaptation functions in this bacterium. The contribution of these three Csps in the host cell invasion processes of L. monocytogenes was investigated based on human Caco-2 and murine macrophage in vitro cell infection models. The DeltacspB, DeltacspD, DeltacspAB, DeltacspAD, DeltacspBD, and DeltacspABD strains were all significantly impaired in Caco-2 cell invasion compared with the wild-type strain, whereas in the murine macrophage infection assay only, the double (DeltacspBD) and triple (DeltacspABD) csp mutants were also significantly impaired in cell invasion compared with the wild-type strain. The DeltacspBD and DeltacspABD mutants displayed the most severely impaired invasion phenotypes. The invasion ability of these two mutant strains was also further analyzed using cold-stress-exposed organisms. In both cell infection models a significant reduction in invasiveness was observed after cold stress exposure of Listeria organisms. The negative impact of cold stress on subsequent cell invasion ability was, however, more severe in cold-sensitive csp mutants (DeltacspBD and DeltacspABD) compared with the wild type. The impaired macrophage invasion and intracellular growth of DeltacspBD and DeltacspABD also led us to examine oxidative stress resistance capacity in these two mutant strains. Both strains also displayed higher oxidative stress sensitivity relative to the wild-type strain. Our data indicate that besides cold and osmotic stress adaptation roles, Csp family proteins also promote efficient host cell invasion and oxidative stress adaptation processes in L. monocytogenes.

Listeria monocytogenes are important foodborne pathogens that can cause outbreaks of serious human disease. These organisms frequently colonize and proliferate on preserved food products despite exposure to stress conditions induced by low storage temperatures, inclusion of organic acid-based preservatives, and high osmolarity. To assess alternative sigma factor sigma(L) contributions to such stress resistance of L. monocytogenes, quantitative RT-PCR assays and sigL gene deletion mutagenesis were applied in L. monocytogenes EGDe. Transcription of sigL was significantly induced by growth of EGDe under cold, organic acid, and elevated NaCl salt concentration stress conditions. The growth of a DeltasigL strain exposed to these stress conditions was also found to be significantly impaired in comparison to that of its isogenic wild-type strain. The contribution of sigma(L) to transcription control of cold and NaCl stress adaptation genes, oppA, cspD, and clpP, was also comparatively assessed in DeltasigL and wild-type EGDe cells. Transcription of the oppA gene, which encodes the OppA protein that also promotes L. monocytogenes cold growth, was significantly reduced in cold stress-grown DeltasigL cells compared to levels of the wild-type EGDe strain. These findings therefore suggest important roles of sigma(L) regulatory pathways in facilitating resistance of L. monocytogenes organisms against stress conditions associated with low storage temperatures, exposure to organic acid, and elevated NaCl salt concentrations.