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Cyclic di-GMP-dependent signaling pathways in the pathogenic Firmicute Listeria monocytogenes.

Chen LH, Köseoğlu VK, Güvener ZT, Myers-Morales T, Reed JM, D'Orazio SE, Miller KW, Gomelsky M - PLoS Pathog. (2014)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, United States of America.

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.

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In vitro assay of c-di-GMP binding by the PssE receptor.A: The MBP-PssE protein purified via affinity (amylose resin) chromatography. The GGDEF domain of PssE (residues 107-285) containing the putative I-site was fused downstream of MBP, MBP::GGDEFpssE, and used in c-di-GMP binding assays. B: Saturation plot of equilibrium binding of c-di-GMP to the PssE receptor (MBP::GGDEFpssE). Shown is the dependence of the ratio of bound c-di-GMP per protein in the dialysis chamber, where protein alone was loaded, versus concentration of free c-di-GMP at equilibrium.
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ppat-1004301-g005: In vitro assay of c-di-GMP binding by the PssE receptor.A: The MBP-PssE protein purified via affinity (amylose resin) chromatography. The GGDEF domain of PssE (residues 107-285) containing the putative I-site was fused downstream of MBP, MBP::GGDEFpssE, and used in c-di-GMP binding assays. B: Saturation plot of equilibrium binding of c-di-GMP to the PssE receptor (MBP::GGDEFpssE). Shown is the dependence of the ratio of bound c-di-GMP per protein in the dialysis chamber, where protein alone was loaded, versus concentration of free c-di-GMP at equilibrium.

Mentions: To test the prediction that the PssE protein acts as a c-di-GMP receptor, we overexpressed its GGDEF domain containing the I-site as an MBP fusion (MBP-GGDEFpssE), purified this protein (Fig. 5A) and analyzed its ability to bind c-di-GMP in vitro using equilibrium dialysis. MBP-GGDEFpssE was found to bind c-di-GMP with an apparent Kd of 0.79±0.17 µM (Fig. 5B). This value falls within the range of physiologically relevant intracellular c-di-GMP concentrations measured in other bacteria that are believed to be in the submicromolar to low micromolar range [5]. The binding capacity of the MBP-GGDEFpssE protein, Bmax, was calculated to be 2.03±0.12 µM c-di-GMP (µM protein)−1 indicating that each PssE molecule can bind two c-di-GMP molecules at saturation. This result is consistent with the observation of an intercalated c-di-GMP dimer bound to the I-sites of crystallized GGDEF domain proteins [2], [3]. Therefore, PssE is a bona fide c-di-GMP receptor that is predicted to transfer the c-di-GMP signal to activate synthesis of the listerial Pss EPS.


Cyclic di-GMP-dependent signaling pathways in the pathogenic Firmicute Listeria monocytogenes.

Chen LH, Köseoğlu VK, Güvener ZT, Myers-Morales T, Reed JM, D'Orazio SE, Miller KW, Gomelsky M - PLoS Pathog. (2014)

In vitro assay of c-di-GMP binding by the PssE receptor.A: The MBP-PssE protein purified via affinity (amylose resin) chromatography. The GGDEF domain of PssE (residues 107-285) containing the putative I-site was fused downstream of MBP, MBP::GGDEFpssE, and used in c-di-GMP binding assays. B: Saturation plot of equilibrium binding of c-di-GMP to the PssE receptor (MBP::GGDEFpssE). Shown is the dependence of the ratio of bound c-di-GMP per protein in the dialysis chamber, where protein alone was loaded, versus concentration of free c-di-GMP at equilibrium.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125290&req=5

ppat-1004301-g005: In vitro assay of c-di-GMP binding by the PssE receptor.A: The MBP-PssE protein purified via affinity (amylose resin) chromatography. The GGDEF domain of PssE (residues 107-285) containing the putative I-site was fused downstream of MBP, MBP::GGDEFpssE, and used in c-di-GMP binding assays. B: Saturation plot of equilibrium binding of c-di-GMP to the PssE receptor (MBP::GGDEFpssE). Shown is the dependence of the ratio of bound c-di-GMP per protein in the dialysis chamber, where protein alone was loaded, versus concentration of free c-di-GMP at equilibrium.
Mentions: To test the prediction that the PssE protein acts as a c-di-GMP receptor, we overexpressed its GGDEF domain containing the I-site as an MBP fusion (MBP-GGDEFpssE), purified this protein (Fig. 5A) and analyzed its ability to bind c-di-GMP in vitro using equilibrium dialysis. MBP-GGDEFpssE was found to bind c-di-GMP with an apparent Kd of 0.79±0.17 µM (Fig. 5B). This value falls within the range of physiologically relevant intracellular c-di-GMP concentrations measured in other bacteria that are believed to be in the submicromolar to low micromolar range [5]. The binding capacity of the MBP-GGDEFpssE protein, Bmax, was calculated to be 2.03±0.12 µM c-di-GMP (µM protein)−1 indicating that each PssE molecule can bind two c-di-GMP molecules at saturation. This result is consistent with the observation of an intercalated c-di-GMP dimer bound to the I-sites of crystallized GGDEF domain proteins [2], [3]. Therefore, PssE is a bona fide c-di-GMP receptor that is predicted to transfer the c-di-GMP signal to activate synthesis of the listerial Pss EPS.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, United States of America.

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.

Show MeSH
Related in: MedlinePlus