We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.

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Published in PLOS Pathogens, v. 10, issue. 8, e1004301.

© 2014 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Figure_S1.pdf (512 kB)
Congo red staining of EPS in the L. monocytogenes pde mutants. Congo red staining shows partially redundant functions of PDEs. Presence of at least one PDE is sufficient to prevent full-scale induction of the EPS synthesis. 1, WT, wild type; 2, ΔpdeB/C/D; 3, ΔpdeB/C; 4, ΔpdeC/D; 5, ΔpdeB/D; 6, ΔpdeD; 7, ΔpdeB; 8, ΔpdeC.

Figure_S2.pdf (65 kB)
Effects of c-di-GMP on intravenous L. monocytogenes infections. Cyclic di-GMP levels do not affect growth in the liver and spleen of L. monocytogenes delivered intravenously. A: Female BALB/c/By/J mice (n = 4) were co-infected intravenously with a 1:1 mixture of wild type made chloramphenicol-resistant (CmR) by chromosomal insertion of pAD1-cYFP (Table 1) and ΔpdeB/C/D mutant (~600 CFU of each for a total inoculum of 1.2×103 CFU). Three days post-infection, spleens and livers were harvested aseptically, homogenized, diluted and plated on BHI agar with or without the presence of 7 µg/ml of chloramphenicol. The number of chloramphenicol-sensitive (CmS) ΔpdeB/C/D CFU was determined by subtracting the number of (CmR) colonies from the total CFU found on plates without antibiotic. Competitive index (CI) ratios were determined by dividing the number of CmS ΔpdeB/C/D CFU by the number of CmR wild type CFU recovered from each tissue. B: A competition experiment performed with the CmR wild type and the strain expressing the E. coli PDE, YhjH. WT, chloramphenicol-resistant (CmR) derivative of strain EGD-e; pIMK::yhjH, EGD-e with integrated plasmid pIMK2 expressing E. coli PDE, YhjH (Table 1).

Table_S1.doc (40 kB)
Primers used in this study.