Limits...
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.

Show MeSH

Related in: MedlinePlus

Impaired invasion of L. monocytogenes in HT-29 human colon adenocarcinoma cells by elevated c-di-GMP levels.A: Expression of the heterologous DGC, Slr1143 (WT::slr; blue bar), or deletion of the native PDEs (ΔpdeB/C/D; black), strongly inhibit listerial invasion, compared to EGD-e containing an empty vector (WT::pIMK; white), while overexpression of the heterologous PDE, YhjH (WT::yhjH; yellow), improves invasion. B: High intracellular c-di-GMP levels inhibit invasion more significantly than the presence of EPS. Strains shown are WT (white bar); ΔpdeB/C/D mutant (black); ΔpdeB/C/D ΔpssC (dark-grey) and ΔpdeB/C/D ΔpssE (light-grey). Plotted are values of relative invasion, compared to those of WT::pIMK (panel A) or WT (panel B). Average results from three independent tests, each performed in three replicates are shown. *, significantly different (p<0.001). Prism 5 for Mac (GraphPad) was used to perform unpaired Student's t-tests.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125290&req=5

ppat-1004301-g007: Impaired invasion of L. monocytogenes in HT-29 human colon adenocarcinoma cells by elevated c-di-GMP levels.A: Expression of the heterologous DGC, Slr1143 (WT::slr; blue bar), or deletion of the native PDEs (ΔpdeB/C/D; black), strongly inhibit listerial invasion, compared to EGD-e containing an empty vector (WT::pIMK; white), while overexpression of the heterologous PDE, YhjH (WT::yhjH; yellow), improves invasion. B: High intracellular c-di-GMP levels inhibit invasion more significantly than the presence of EPS. Strains shown are WT (white bar); ΔpdeB/C/D mutant (black); ΔpdeB/C/D ΔpssC (dark-grey) and ΔpdeB/C/D ΔpssE (light-grey). Plotted are values of relative invasion, compared to those of WT::pIMK (panel A) or WT (panel B). Average results from three independent tests, each performed in three replicates are shown. *, significantly different (p<0.001). Prism 5 for Mac (GraphPad) was used to perform unpaired Student's t-tests.

Mentions: As a foodborne pathogen, L. monocytogenes is expected to use gut epithelial cells for primary invasion [17]–[20]. We examined the consequences of elevated c-di-GMP levels on bacterial invasion into HT-29 human colon adenocarcinoma cells. As shown in Fig. 7A, the strains with elevated c-di-GMP levels were significantly impaired in invasion, whether elevated c-di-GMP was caused by expression of the heterologous DGC, Slr1143, or by the ΔpdeB/C/D mutations. Consistent with the inhibitory role of c-di-GMP, invasion was increased, by approximately 2-fold, in the L. monocytogenes strain expressing a c-di-GMP PDE, YhjH.


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)

Impaired invasion of L. monocytogenes in HT-29 human colon adenocarcinoma cells by elevated c-di-GMP levels.A: Expression of the heterologous DGC, Slr1143 (WT::slr; blue bar), or deletion of the native PDEs (ΔpdeB/C/D; black), strongly inhibit listerial invasion, compared to EGD-e containing an empty vector (WT::pIMK; white), while overexpression of the heterologous PDE, YhjH (WT::yhjH; yellow), improves invasion. B: High intracellular c-di-GMP levels inhibit invasion more significantly than the presence of EPS. Strains shown are WT (white bar); ΔpdeB/C/D mutant (black); ΔpdeB/C/D ΔpssC (dark-grey) and ΔpdeB/C/D ΔpssE (light-grey). Plotted are values of relative invasion, compared to those of WT::pIMK (panel A) or WT (panel B). Average results from three independent tests, each performed in three replicates are shown. *, significantly different (p<0.001). Prism 5 for Mac (GraphPad) was used to perform unpaired Student's t-tests.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004301-g007: Impaired invasion of L. monocytogenes in HT-29 human colon adenocarcinoma cells by elevated c-di-GMP levels.A: Expression of the heterologous DGC, Slr1143 (WT::slr; blue bar), or deletion of the native PDEs (ΔpdeB/C/D; black), strongly inhibit listerial invasion, compared to EGD-e containing an empty vector (WT::pIMK; white), while overexpression of the heterologous PDE, YhjH (WT::yhjH; yellow), improves invasion. B: High intracellular c-di-GMP levels inhibit invasion more significantly than the presence of EPS. Strains shown are WT (white bar); ΔpdeB/C/D mutant (black); ΔpdeB/C/D ΔpssC (dark-grey) and ΔpdeB/C/D ΔpssE (light-grey). Plotted are values of relative invasion, compared to those of WT::pIMK (panel A) or WT (panel B). Average results from three independent tests, each performed in three replicates are shown. *, significantly different (p<0.001). Prism 5 for Mac (GraphPad) was used to perform unpaired Student's t-tests.
Mentions: As a foodborne pathogen, L. monocytogenes is expected to use gut epithelial cells for primary invasion [17]–[20]. We examined the consequences of elevated c-di-GMP levels on bacterial invasion into HT-29 human colon adenocarcinoma cells. As shown in Fig. 7A, the strains with elevated c-di-GMP levels were significantly impaired in invasion, whether elevated c-di-GMP was caused by expression of the heterologous DGC, Slr1143, or by the ΔpdeB/C/D mutations. Consistent with the inhibitory role of c-di-GMP, invasion was increased, by approximately 2-fold, in the L. monocytogenes strain expressing a c-di-GMP PDE, YhjH.

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