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

Inhibition of motility and activation of EPS production in L. monocytogenes by elevated levels of c-di-GMP.A: Top, Inhibition of swimming of the wild-type L. monocytogenes in semi-solid agar by a heterologous DGC, Slr1143. Bottom, Restoration of swimming in semi-solid agar of the L. monocytogenes ΔpdeB/C/D mutant by a heterologous PDE, YhjH. WT, wild type, EGD-e; A/B/C, ΔpdeB/C/D mutant; pIMK, WT::pIMK2 (vector control); slr, WT::(pIMK2::slr1143); yhjH, WT::(pIMK2::yhjH). B: Congo red staining of EPS in L. monocytogenes. 1, WT, wild type; 2, ΔpdeB/C/D; 3, ΔpdeB/C/D ΔpssE; 4, ΔpdeB/C/D ΔpssC; 5, ΔpdeB/C/D::pIMK2; 6, ΔpdeB/C/D::pIMK2::yhjH; 7, ΔpdeB/C/D::(pIMK2::slr1143); 8, WT::pIMK2; 9, WT::(pIMK2::yhjH); 10, WT::(pIMK2::slr1143). C: Deletion of all three c-di-GMP PDEs drastically inhibits motility of L. monocytogenes in semi-solid agar. WT, wild type strain, B, ΔpdeB; C, ΔpdeC; D, ΔpdeD; B/D, ΔpdeB ΔpdeD; C/D, ΔpdeC ΔpdeD; B/C, ΔpdeB ΔpdeC; B/C/D, ΔpdeB/C/D. D: Rough colony morphology and increased Congo red staining of the L. monocytogenes ΔpdeB/C/D mutant and rescue of the wild-type colony morphology by the ΔpssC mutation (ΔpdeB/C/D ΔpssC). E: Restoration of motility of the ΔpdeB/C/D mutant by the ΔpssC or ΔpssE mutations.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004301-g004: Inhibition of motility and activation of EPS production in L. monocytogenes by elevated levels of c-di-GMP.A: Top, Inhibition of swimming of the wild-type L. monocytogenes in semi-solid agar by a heterologous DGC, Slr1143. Bottom, Restoration of swimming in semi-solid agar of the L. monocytogenes ΔpdeB/C/D mutant by a heterologous PDE, YhjH. WT, wild type, EGD-e; A/B/C, ΔpdeB/C/D mutant; pIMK, WT::pIMK2 (vector control); slr, WT::(pIMK2::slr1143); yhjH, WT::(pIMK2::yhjH). B: Congo red staining of EPS in L. monocytogenes. 1, WT, wild type; 2, ΔpdeB/C/D; 3, ΔpdeB/C/D ΔpssE; 4, ΔpdeB/C/D ΔpssC; 5, ΔpdeB/C/D::pIMK2; 6, ΔpdeB/C/D::pIMK2::yhjH; 7, ΔpdeB/C/D::(pIMK2::slr1143); 8, WT::pIMK2; 9, WT::(pIMK2::yhjH); 10, WT::(pIMK2::slr1143). C: Deletion of all three c-di-GMP PDEs drastically inhibits motility of L. monocytogenes in semi-solid agar. WT, wild type strain, B, ΔpdeB; C, ΔpdeC; D, ΔpdeD; B/D, ΔpdeB ΔpdeD; C/D, ΔpdeC ΔpdeD; B/C, ΔpdeB ΔpdeC; B/C/D, ΔpdeB/C/D. D: Rough colony morphology and increased Congo red staining of the L. monocytogenes ΔpdeB/C/D mutant and rescue of the wild-type colony morphology by the ΔpssC mutation (ΔpdeB/C/D ΔpssC). E: Restoration of motility of the ΔpdeB/C/D mutant by the ΔpssC or ΔpssE mutations.

Mentions: L. monocytogenes uses flagella for motility [54]. Expression of Slr1143 blocked swimming of strain EGD-e in semi-solid agar, whereas expression of YhjH had no effect (Fig. 4A top). Expression of Slr1143 also resulted in more pigmented L. monocytogenes colonies on Congo red agar, whereas expression of YhjH had no observable phenotype (Fig. 4B, sectors 10 versus 1 and 9). Later in this work we show that YhjH is expressed and functional as a PDE in L. monocytogenes. Therefore, we interpreted the lack of a phenotype associated with YhjH overexpression as an indication that intracellular c-di-GMP levels in strain EGD-e are already low, and that c-di-GMP does not play a significant role under the conditions used in these assays. Since L. monocytogenes is not known to synthesize pili, and the genome of strain EGD-e has no candidate pili genes, we hypothesized that Congo red staining was indicative of EPS production. An EPS has been suspected in some naturally occurring L. monocytogenes isolates [28]. Further, Tiensuu and colleagues have recently observed Congo red staining rings within L. monocytogenes colonies exposed to dark-light cycles [55], however the nature of the Congo red-binding extracellular polymer was not investigated.


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)

Inhibition of motility and activation of EPS production in L. monocytogenes by elevated levels of c-di-GMP.A: Top, Inhibition of swimming of the wild-type L. monocytogenes in semi-solid agar by a heterologous DGC, Slr1143. Bottom, Restoration of swimming in semi-solid agar of the L. monocytogenes ΔpdeB/C/D mutant by a heterologous PDE, YhjH. WT, wild type, EGD-e; A/B/C, ΔpdeB/C/D mutant; pIMK, WT::pIMK2 (vector control); slr, WT::(pIMK2::slr1143); yhjH, WT::(pIMK2::yhjH). B: Congo red staining of EPS in L. monocytogenes. 1, WT, wild type; 2, ΔpdeB/C/D; 3, ΔpdeB/C/D ΔpssE; 4, ΔpdeB/C/D ΔpssC; 5, ΔpdeB/C/D::pIMK2; 6, ΔpdeB/C/D::pIMK2::yhjH; 7, ΔpdeB/C/D::(pIMK2::slr1143); 8, WT::pIMK2; 9, WT::(pIMK2::yhjH); 10, WT::(pIMK2::slr1143). C: Deletion of all three c-di-GMP PDEs drastically inhibits motility of L. monocytogenes in semi-solid agar. WT, wild type strain, B, ΔpdeB; C, ΔpdeC; D, ΔpdeD; B/D, ΔpdeB ΔpdeD; C/D, ΔpdeC ΔpdeD; B/C, ΔpdeB ΔpdeC; B/C/D, ΔpdeB/C/D. D: Rough colony morphology and increased Congo red staining of the L. monocytogenes ΔpdeB/C/D mutant and rescue of the wild-type colony morphology by the ΔpssC mutation (ΔpdeB/C/D ΔpssC). E: Restoration of motility of the ΔpdeB/C/D mutant by the ΔpssC or ΔpssE mutations.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004301-g004: Inhibition of motility and activation of EPS production in L. monocytogenes by elevated levels of c-di-GMP.A: Top, Inhibition of swimming of the wild-type L. monocytogenes in semi-solid agar by a heterologous DGC, Slr1143. Bottom, Restoration of swimming in semi-solid agar of the L. monocytogenes ΔpdeB/C/D mutant by a heterologous PDE, YhjH. WT, wild type, EGD-e; A/B/C, ΔpdeB/C/D mutant; pIMK, WT::pIMK2 (vector control); slr, WT::(pIMK2::slr1143); yhjH, WT::(pIMK2::yhjH). B: Congo red staining of EPS in L. monocytogenes. 1, WT, wild type; 2, ΔpdeB/C/D; 3, ΔpdeB/C/D ΔpssE; 4, ΔpdeB/C/D ΔpssC; 5, ΔpdeB/C/D::pIMK2; 6, ΔpdeB/C/D::pIMK2::yhjH; 7, ΔpdeB/C/D::(pIMK2::slr1143); 8, WT::pIMK2; 9, WT::(pIMK2::yhjH); 10, WT::(pIMK2::slr1143). C: Deletion of all three c-di-GMP PDEs drastically inhibits motility of L. monocytogenes in semi-solid agar. WT, wild type strain, B, ΔpdeB; C, ΔpdeC; D, ΔpdeD; B/D, ΔpdeB ΔpdeD; C/D, ΔpdeC ΔpdeD; B/C, ΔpdeB ΔpdeC; B/C/D, ΔpdeB/C/D. D: Rough colony morphology and increased Congo red staining of the L. monocytogenes ΔpdeB/C/D mutant and rescue of the wild-type colony morphology by the ΔpssC mutation (ΔpdeB/C/D ΔpssC). E: Restoration of motility of the ΔpdeB/C/D mutant by the ΔpssC or ΔpssE mutations.
Mentions: L. monocytogenes uses flagella for motility [54]. Expression of Slr1143 blocked swimming of strain EGD-e in semi-solid agar, whereas expression of YhjH had no effect (Fig. 4A top). Expression of Slr1143 also resulted in more pigmented L. monocytogenes colonies on Congo red agar, whereas expression of YhjH had no observable phenotype (Fig. 4B, sectors 10 versus 1 and 9). Later in this work we show that YhjH is expressed and functional as a PDE in L. monocytogenes. Therefore, we interpreted the lack of a phenotype associated with YhjH overexpression as an indication that intracellular c-di-GMP levels in strain EGD-e are already low, and that c-di-GMP does not play a significant role under the conditions used in these assays. Since L. monocytogenes is not known to synthesize pili, and the genome of strain EGD-e has no candidate pili genes, we hypothesized that Congo red staining was indicative of EPS production. An EPS has been suspected in some naturally occurring L. monocytogenes isolates [28]. Further, Tiensuu and colleagues have recently observed Congo red staining rings within L. monocytogenes colonies exposed to dark-light cycles [55], however the nature of the Congo red-binding extracellular polymer was not investigated.

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