Abstract
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.
Highlights
Cyclic dimeric GMP (c-di-GMP) [1] is one of the most common bacterial second messengers
C-di-GMP is synthesized by diguanylate cyclases (DGCs), which contain GGDEF domains [36,37], and degraded by c-di-GMPspecific phosphodiesterases (PDEs), which contain either EAL [38,39,40] or HD-GYP [41] catalytic domains
The three predicted DGCs have similar domain architectures with a GGDEF domain preceded by either six or eight transmembrane helices (Fig. 1). This domain architecture suggests that c-di-GMP synthesis is regulated by external signals or signals derived from the cell wall or cytoplasmic membrane
Summary
Cyclic dimeric GMP (c-di-GMP) [1] is one of the most common bacterial second messengers. Over the last ten years our understanding of c-di-GMP-mediated signal transduction pathways has rapidly expanded (reviewed in [2,3,4,5,6]). This expansion has been dominated by studies of Proteobacteria, and to a lesser extent Actinobacteria and Spirochetes, while studies of cdi-GMP signaling in Firmicutes have lagged behind. In pathogens that propagate extracellularly, elevated c-di-GMP levels have been found generally detrimental for acute infections (reviewed in [2,5,6,7]), individual components of c-di-GMP signaling networks may play different roles during various stages of infection [8,9]. C-di-GMP signaling pathways are required for full-scale virulence in those species that form biofilm-like intracellular structures [13,14,15], but appear to be detrimental, at least in some species, that do not form such structures [16]
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