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The host protein calprotectin modulates the Helicobacter pylori cag type IV secretion system via zinc sequestration.

Gaddy JA, Radin JN, Loh JT, Piazuelo MB, Kehl-Fie TE, Delgado AG, Ilca FT, Peek RM, Cover TL, Chazin WJ, Skaar EP, Scott Algood HM - PLoS Pathog. (2014)

Bottom Line: When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice.In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion.Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.

View Article: PubMed Central - PubMed

Affiliation: Veterans Affairs Tennessee Valley Healthcare Services, Nashville, Tennessee, United States of America; Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.

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H. pylori cag T4SS pilus production is modulated by the zinc sequestration activity of calprotectin.High resolution FE-SEM analysis of H. pylori co-cultured with AGS human gastric epithelial cells. Bacteria were cultured in medium alone, or medium supplemented with wild-type CP (200 µg/mL) or mutant forms of CP that lack S1, S2 or both metal-binding sites (DS) at 200 µg/mL, 600 µg/mL, or 1200 µg/mL. Pilus formation was also examined using the synthetic zinc chelator, TPEN. Supplementation with 100 µM zinc chloride (+Zinc) restored T4SS pilus formation to levels comparable to medium alone in all samples. Arrows indicate cag T4SS pili formed at the host-pathogen interface. Magnification bars indicate 1 µm. Dot plot graphs indicate enumeration of pili per bacterial cell as quantified from representative micrographs derived from three biological replicates, and at least 20 fields from each replicate. Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to first condition on each graph (Student's t test).
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ppat-1004450-g006: H. pylori cag T4SS pilus production is modulated by the zinc sequestration activity of calprotectin.High resolution FE-SEM analysis of H. pylori co-cultured with AGS human gastric epithelial cells. Bacteria were cultured in medium alone, or medium supplemented with wild-type CP (200 µg/mL) or mutant forms of CP that lack S1, S2 or both metal-binding sites (DS) at 200 µg/mL, 600 µg/mL, or 1200 µg/mL. Pilus formation was also examined using the synthetic zinc chelator, TPEN. Supplementation with 100 µM zinc chloride (+Zinc) restored T4SS pilus formation to levels comparable to medium alone in all samples. Arrows indicate cag T4SS pili formed at the host-pathogen interface. Magnification bars indicate 1 µm. Dot plot graphs indicate enumeration of pili per bacterial cell as quantified from representative micrographs derived from three biological replicates, and at least 20 fields from each replicate. Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to first condition on each graph (Student's t test).

Mentions: Our co-culture experiments demonstrated that metal sequestration by CP leads to abrogated phosphorylation of CagA and inhibition of downstream cellular activation in gastric epithelial cells. We next tested the hypothesis that the observed inhibition of the cag T4SS-dependent phenotype was attributable to inhibition of cag T4SS pilus production. To test this, field emission gun-scanning electron microscopy (FEG-SEM) analysis of the bacterial-gastric epithelial cell (H. pylori-AGS cell) co-cultures was performed to visualize the cag T4SS pili, as previously described [25], [32], [34]. Briefly, bacteria were cultured for 4–6 hours prior to co-culture with AGS cells in the presence or absence of WT or mutant forms of CP or the synthetic zinc chelator, TPEN, alone or in the presence of an exogenous source of nutrient zinc. Bacteria were co-cultured with gastric cells in the absence of additives for 4 hours before samples were processed and analyzed by high resolution FEG-SEM. Pre-treatment of H. pylori with CP or TPEN reduced the number of cag T4SS pili visible at the host pathogen interface (Figure 6). The addition of exogenous zinc restored WT pili formation, suggesting that the zinc-chelation is responsible for this reduced pilus formation. Similarly, the ΔS1 and ΔS2 mutant CP proteins were unable to repress pilus formation at the same concentration as WT (200 µg/mL). However, when increasing the concentration of the ΔS1 and ΔS2 mutants to high concentrations (600 µg/ml), cag T4SS pilus formation was repressed. The higher concentration of both ΔS1 and ΔS2 mutant CP proteins was consistent with earlier results, where 600 µg/ml of these mutant proteins was necessary to observe decreased NFkB and IL-8 expression. Conversely, the DS mutant CP did not repress cag T4SS pilus formation, even at very high concentrations (1200 µg/ml). These data indicate that CP inhibits the production of cag T4SS-associated pili through zinc sequestration.


The host protein calprotectin modulates the Helicobacter pylori cag type IV secretion system via zinc sequestration.

Gaddy JA, Radin JN, Loh JT, Piazuelo MB, Kehl-Fie TE, Delgado AG, Ilca FT, Peek RM, Cover TL, Chazin WJ, Skaar EP, Scott Algood HM - PLoS Pathog. (2014)

H. pylori cag T4SS pilus production is modulated by the zinc sequestration activity of calprotectin.High resolution FE-SEM analysis of H. pylori co-cultured with AGS human gastric epithelial cells. Bacteria were cultured in medium alone, or medium supplemented with wild-type CP (200 µg/mL) or mutant forms of CP that lack S1, S2 or both metal-binding sites (DS) at 200 µg/mL, 600 µg/mL, or 1200 µg/mL. Pilus formation was also examined using the synthetic zinc chelator, TPEN. Supplementation with 100 µM zinc chloride (+Zinc) restored T4SS pilus formation to levels comparable to medium alone in all samples. Arrows indicate cag T4SS pili formed at the host-pathogen interface. Magnification bars indicate 1 µm. Dot plot graphs indicate enumeration of pili per bacterial cell as quantified from representative micrographs derived from three biological replicates, and at least 20 fields from each replicate. Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to first condition on each graph (Student's t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199781&req=5

ppat-1004450-g006: H. pylori cag T4SS pilus production is modulated by the zinc sequestration activity of calprotectin.High resolution FE-SEM analysis of H. pylori co-cultured with AGS human gastric epithelial cells. Bacteria were cultured in medium alone, or medium supplemented with wild-type CP (200 µg/mL) or mutant forms of CP that lack S1, S2 or both metal-binding sites (DS) at 200 µg/mL, 600 µg/mL, or 1200 µg/mL. Pilus formation was also examined using the synthetic zinc chelator, TPEN. Supplementation with 100 µM zinc chloride (+Zinc) restored T4SS pilus formation to levels comparable to medium alone in all samples. Arrows indicate cag T4SS pili formed at the host-pathogen interface. Magnification bars indicate 1 µm. Dot plot graphs indicate enumeration of pili per bacterial cell as quantified from representative micrographs derived from three biological replicates, and at least 20 fields from each replicate. Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to first condition on each graph (Student's t test).
Mentions: Our co-culture experiments demonstrated that metal sequestration by CP leads to abrogated phosphorylation of CagA and inhibition of downstream cellular activation in gastric epithelial cells. We next tested the hypothesis that the observed inhibition of the cag T4SS-dependent phenotype was attributable to inhibition of cag T4SS pilus production. To test this, field emission gun-scanning electron microscopy (FEG-SEM) analysis of the bacterial-gastric epithelial cell (H. pylori-AGS cell) co-cultures was performed to visualize the cag T4SS pili, as previously described [25], [32], [34]. Briefly, bacteria were cultured for 4–6 hours prior to co-culture with AGS cells in the presence or absence of WT or mutant forms of CP or the synthetic zinc chelator, TPEN, alone or in the presence of an exogenous source of nutrient zinc. Bacteria were co-cultured with gastric cells in the absence of additives for 4 hours before samples were processed and analyzed by high resolution FEG-SEM. Pre-treatment of H. pylori with CP or TPEN reduced the number of cag T4SS pili visible at the host pathogen interface (Figure 6). The addition of exogenous zinc restored WT pili formation, suggesting that the zinc-chelation is responsible for this reduced pilus formation. Similarly, the ΔS1 and ΔS2 mutant CP proteins were unable to repress pilus formation at the same concentration as WT (200 µg/mL). However, when increasing the concentration of the ΔS1 and ΔS2 mutants to high concentrations (600 µg/ml), cag T4SS pilus formation was repressed. The higher concentration of both ΔS1 and ΔS2 mutant CP proteins was consistent with earlier results, where 600 µg/ml of these mutant proteins was necessary to observe decreased NFkB and IL-8 expression. Conversely, the DS mutant CP did not repress cag T4SS pilus formation, even at very high concentrations (1200 µg/ml). These data indicate that CP inhibits the production of cag T4SS-associated pili through zinc sequestration.

Bottom Line: When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice.In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion.Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.

View Article: PubMed Central - PubMed

Affiliation: Veterans Affairs Tennessee Valley Healthcare Services, Nashville, Tennessee, United States of America; Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.

Show MeSH
Related in: MedlinePlus