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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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NFκB activation and IL-8 secretion in response to co-culture with H. pylori is dependent on zinc availability.Bacteria were grown in medium alone or medium supplemented with the synthetic zinc chelator TPEN (TPEN), wild-type CP (CP) at 200 µg/mL, or mutant forms of CP [ΔS1 or ΔS2 at 200 µg/mL or 600 µg/mL, or a double-site mutant (DS) at 1200 µg/mL] alone or in the presence of 100 µM zinc chloride (+Zinc) prior to co-culture with AGS cells. A) NFκB activation in human gastric epithelial cells co-cultured with H. pylori for 4 hours was quantified using a luciferase reporter assay. B) IL-8 secretion by human gastric epithelial cells was quantified using an IL-8 ELISA assay. Bars represent the mean +/− SEM per each group (n = 3–5 biological replicates). Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to medium alone (Student's t test).
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ppat-1004450-g005: NFκB activation and IL-8 secretion in response to co-culture with H. pylori is dependent on zinc availability.Bacteria were grown in medium alone or medium supplemented with the synthetic zinc chelator TPEN (TPEN), wild-type CP (CP) at 200 µg/mL, or mutant forms of CP [ΔS1 or ΔS2 at 200 µg/mL or 600 µg/mL, or a double-site mutant (DS) at 1200 µg/mL] alone or in the presence of 100 µM zinc chloride (+Zinc) prior to co-culture with AGS cells. A) NFκB activation in human gastric epithelial cells co-cultured with H. pylori for 4 hours was quantified using a luciferase reporter assay. B) IL-8 secretion by human gastric epithelial cells was quantified using an IL-8 ELISA assay. Bars represent the mean +/− SEM per each group (n = 3–5 biological replicates). Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to medium alone (Student's t test).

Mentions: A functional cag T4SS translocates the effector molecule, CagA, into host cells, where it is then phosphorylated [25]. Moreover, a functional cag T4SS is necessary for activation of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) in human AGS gastric epithelial cells, which is a result of both CagA translocation and peptidoglycan recognition by NOD1 [33]. As a result of these cellular signaling events, IL-8 is produced and secreted by the AGS gastric epithelial cells. Therefore, the functional activity of the cag T4SS was measured with three assays; CagA translocation and phosphorylation (Figure 4), NFκB activation (Figure 5A), and IL-8 secretion by gastric epithelial cells (Figure 5B).


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)

NFκB activation and IL-8 secretion in response to co-culture with H. pylori is dependent on zinc availability.Bacteria were grown in medium alone or medium supplemented with the synthetic zinc chelator TPEN (TPEN), wild-type CP (CP) at 200 µg/mL, or mutant forms of CP [ΔS1 or ΔS2 at 200 µg/mL or 600 µg/mL, or a double-site mutant (DS) at 1200 µg/mL] alone or in the presence of 100 µM zinc chloride (+Zinc) prior to co-culture with AGS cells. A) NFκB activation in human gastric epithelial cells co-cultured with H. pylori for 4 hours was quantified using a luciferase reporter assay. B) IL-8 secretion by human gastric epithelial cells was quantified using an IL-8 ELISA assay. Bars represent the mean +/− SEM per each group (n = 3–5 biological replicates). Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to medium alone (Student's t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199781&req=5

ppat-1004450-g005: NFκB activation and IL-8 secretion in response to co-culture with H. pylori is dependent on zinc availability.Bacteria were grown in medium alone or medium supplemented with the synthetic zinc chelator TPEN (TPEN), wild-type CP (CP) at 200 µg/mL, or mutant forms of CP [ΔS1 or ΔS2 at 200 µg/mL or 600 µg/mL, or a double-site mutant (DS) at 1200 µg/mL] alone or in the presence of 100 µM zinc chloride (+Zinc) prior to co-culture with AGS cells. A) NFκB activation in human gastric epithelial cells co-cultured with H. pylori for 4 hours was quantified using a luciferase reporter assay. B) IL-8 secretion by human gastric epithelial cells was quantified using an IL-8 ELISA assay. Bars represent the mean +/− SEM per each group (n = 3–5 biological replicates). Asterisks indicate *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared to medium alone (Student's t test).
Mentions: A functional cag T4SS translocates the effector molecule, CagA, into host cells, where it is then phosphorylated [25]. Moreover, a functional cag T4SS is necessary for activation of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) in human AGS gastric epithelial cells, which is a result of both CagA translocation and peptidoglycan recognition by NOD1 [33]. As a result of these cellular signaling events, IL-8 is produced and secreted by the AGS gastric epithelial cells. Therefore, the functional activity of the cag T4SS was measured with three assays; CagA translocation and phosphorylation (Figure 4), NFκB activation (Figure 5A), and IL-8 secretion by gastric epithelial cells (Figure 5B).

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