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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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PDE activities of the L. monocytogenes proteins PdeB-D.A: Restoration of motility in semi-solid (0.25%) agar of strain MG1655 ΔyhjH by L. monocytogenes PdeB, PdeC and PdeD is indicative of their c-di-GMP PDE activities. PdeB-D were expressed as C-terminal His6-fusions downstream of the T7 promoter from vector pET23a. Although MG1655 does not encode a T7 RNA polymerase gene, the pde genes were expressed from a fortuitous promoter at sufficiently high levels to partially restore the swimming defect of MG1655 ΔyhjH in semi-solid agar. pET, empty vector (pET23a). B: Affinity purified L. monocytogenes PdeD (PdeD::His6), PdeB (PdeB::His6) and PdeC (MBP::PdeC) proteins used in the PDE assays. MW, molecular weight, kD. C: PDE activities of PdeD::His6, PdeB::His6 and MBP::PdeC monitored by the rates of formation of pGpG, the product of c-di-GMP hydrolysis. Nucleotides were measured by HPLC as described earlier [38].
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ppat-1004301-g002: PDE activities of the L. monocytogenes proteins PdeB-D.A: Restoration of motility in semi-solid (0.25%) agar of strain MG1655 ΔyhjH by L. monocytogenes PdeB, PdeC and PdeD is indicative of their c-di-GMP PDE activities. PdeB-D were expressed as C-terminal His6-fusions downstream of the T7 promoter from vector pET23a. Although MG1655 does not encode a T7 RNA polymerase gene, the pde genes were expressed from a fortuitous promoter at sufficiently high levels to partially restore the swimming defect of MG1655 ΔyhjH in semi-solid agar. pET, empty vector (pET23a). B: Affinity purified L. monocytogenes PdeD (PdeD::His6), PdeB (PdeB::His6) and PdeC (MBP::PdeC) proteins used in the PDE assays. MW, molecular weight, kD. C: PDE activities of PdeD::His6, PdeB::His6 and MBP::PdeC monitored by the rates of formation of pGpG, the product of c-di-GMP hydrolysis. Nucleotides were measured by HPLC as described earlier [38].

Mentions: We expressed L. monocytogenes pdeB, pdeC and pdeD in E. coli MG1655 ΔyhjH. This mutant lacks a major c-di-GMP PDE, YhjH [50], [51], and as a result, is impaired in motility in semi-solid agar [52]. We found that expression of any one of the pde genes was sufficient to partially restore swim zones of MG1655 ΔyhjH in semi-solid agar (Fig. 2A). These results are consistent with the possibility that all three proteins, PdeB, PdeC, and PdeD, function as c-di-GMP PDEs. However, overexpressed but enzymatically inactive EAL domain proteins that retain the ability to bind (but not to hydrolyze) c-di-GMP also can lower intracellular c-di-GMP concentration thus mimicking the phenotypes of overexpressed PDEs [53].


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)

PDE activities of the L. monocytogenes proteins PdeB-D.A: Restoration of motility in semi-solid (0.25%) agar of strain MG1655 ΔyhjH by L. monocytogenes PdeB, PdeC and PdeD is indicative of their c-di-GMP PDE activities. PdeB-D were expressed as C-terminal His6-fusions downstream of the T7 promoter from vector pET23a. Although MG1655 does not encode a T7 RNA polymerase gene, the pde genes were expressed from a fortuitous promoter at sufficiently high levels to partially restore the swimming defect of MG1655 ΔyhjH in semi-solid agar. pET, empty vector (pET23a). B: Affinity purified L. monocytogenes PdeD (PdeD::His6), PdeB (PdeB::His6) and PdeC (MBP::PdeC) proteins used in the PDE assays. MW, molecular weight, kD. C: PDE activities of PdeD::His6, PdeB::His6 and MBP::PdeC monitored by the rates of formation of pGpG, the product of c-di-GMP hydrolysis. Nucleotides were measured by HPLC as described earlier [38].
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004301-g002: PDE activities of the L. monocytogenes proteins PdeB-D.A: Restoration of motility in semi-solid (0.25%) agar of strain MG1655 ΔyhjH by L. monocytogenes PdeB, PdeC and PdeD is indicative of their c-di-GMP PDE activities. PdeB-D were expressed as C-terminal His6-fusions downstream of the T7 promoter from vector pET23a. Although MG1655 does not encode a T7 RNA polymerase gene, the pde genes were expressed from a fortuitous promoter at sufficiently high levels to partially restore the swimming defect of MG1655 ΔyhjH in semi-solid agar. pET, empty vector (pET23a). B: Affinity purified L. monocytogenes PdeD (PdeD::His6), PdeB (PdeB::His6) and PdeC (MBP::PdeC) proteins used in the PDE assays. MW, molecular weight, kD. C: PDE activities of PdeD::His6, PdeB::His6 and MBP::PdeC monitored by the rates of formation of pGpG, the product of c-di-GMP hydrolysis. Nucleotides were measured by HPLC as described earlier [38].
Mentions: We expressed L. monocytogenes pdeB, pdeC and pdeD in E. coli MG1655 ΔyhjH. This mutant lacks a major c-di-GMP PDE, YhjH [50], [51], and as a result, is impaired in motility in semi-solid agar [52]. We found that expression of any one of the pde genes was sufficient to partially restore swim zones of MG1655 ΔyhjH in semi-solid agar (Fig. 2A). These results are consistent with the possibility that all three proteins, PdeB, PdeC, and PdeD, function as c-di-GMP PDEs. However, overexpressed but enzymatically inactive EAL domain proteins that retain the ability to bind (but not to hydrolyze) c-di-GMP also can lower intracellular c-di-GMP concentration thus mimicking the phenotypes of overexpressed PDEs [53].

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