The role of the bacterial signaling molecule c-di-GMP in the environmental lifestyle of the food-borne pathogen Listeria monocytogenes

Listeria monocytogenes is the etiological agent of the food-borne infection listeriosis, which primarily affects immunocompromised individuals, the elderly, pregnant women and newborns due to ingestion of contaminated food. The ability of L. monocytogenes to persist in environmental settings increases the risk for its contamination of food products. The bacterial signaling molecule cyclic-di-GMP (c-di-GMP) regulates various features of bacterial physiology, especially the enhancement of biofilm formation via induction of adhesive cell surface structures such as fimbria and exopolysaccharides (EPS). Intracellular c-di-GMP levels are controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs); the former enzymes synthesize c-di-GMP while the letter enzymes hydrolyze it. Finally, bacterial cells contain c-di-GMP binding receptors/effectors that exert the regulatory functions of this signaling molecule. In this study, we investigated the role of c-di-GMP signaling in the Gram-positive intracellular pathogen L. monocytogenes.;Bioinformatics and biochemical studies performed in our laboratories showed that c-di-GMP signaling proteins are present and functional in L. monocytogenes. An elevated level ofc-di-GMP caused by deletion of PDEs induces EPS synthesis in L. monocytogenes via binding to a receptor (PssE), which resides in the poly saccharide synthesis operon (pssA-E ). Rather than playing a role in pathogenesis, c-di-GMP-regulated EPS seems to be involved in the environmental lifestyle of Listeria . EPS overproduction leads to cell aggregation. Listerial cells embedded in these aggregates are remarkably protected against several disinfectants and long-term desiccation. Therefore, c-di-GMP regulation of the novel listerial EPS emerges as a strategy for L. monocytogenes to persist in the environment.;Structural characterization studies revealed that listerial EPS is composed of N-acetylmannosamine (ManNAc) and galactose (Gal). Genetic characterization of EPS biosynthesis showed that the pssA-E operon genes are necessary for EPS production. In addition, a glycosylhydrolase (PssZ) is also required for optimum EPS production. PssZ is located in a c-di-GMP signaling module that consists of the dgcA, dgcB, pssZ and pdeC genes. The diguanylate cyclases, DgcA and DgcB contribute to the c-di-GMP pool used to activate EPS biosynthesis. PssZ can prevent cell aggregation and disperse cell aggregates via degrading cell-surface associated EPS. Overall, the composition of listerial EPS and components of its biosynthetic machinery were determined along with the function of the dedicated glycosylhydrolase. PssZ has the potential to be employed against environmental listerial aggregates and probably against other pathogens belonging to firmicutes that also harbor pssA-E homologs. Indeed, excess PssZ not only disperses cell aggregates but also reduces biofilm formation on surfaces.;The regulation of EPS biosynthesis was also examined in this study. RT-qPCR experiments demonstrated that c-di-GMP is not involved in transcriptional regulation of the pssA-E operon. We also investigated environmental signals that trigger c-di-GMP synthesis and therefore EPS production. High temperature and anaerobic conditions were shown to be detrimental for EPS production. In addition, rich nutrient composition (i.e., addition of brain-heart infusion (BHI) powder) is also inhibitory for EPS production due to the reduction of intracellular c-di-GMP concentration. We showed that the BHI fraction smaller than 10 kDa causes dispersion of cell aggregates and reduced EPS production which are associated with the decreased level of c-di-GMP. Therefore, it becomes important to decipher the BHI components that decrease the activity of DgcA and DgcB. In this regard, the lmo1910 gene residing right upstream of the c-di-GMP signaling module is a strong candidate for regulation of these cyclases. It encodes a protein similar to oxidoreductases, which perform oxidation of neutral and basic D-amino acids. Thus, these types of amino acids might be participating in regulation of DgcA and DgcB activity via Lmo1910.;In summary, the ManNAc-Gal EPS can contribute to survival and persistence of L. monocytogenes in non-host environments. High intracellular c-di-GMP activates the EPS biosynthetic machinery, thus the environmental cues sensed by listerial DGCs and PDEs need to be determined. Finally, L. monocytogenes provides a control agent, the dedicated hydrolase PssZ that can potentially be applied to prevent listerial cell aggregation in food production plants.