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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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Inhibition of H. pylori growth in vitro by CP or TPEN is dose dependent.A) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 50 µM zinc chloride plus 50 µM manganese chloride (Medium+Zinc+Manganese) and with increasing concentrations of CP. B) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 100 µM zinc chloride (Medium+Zinc) and with increasing concentrations of TPEN, a synthetic zinc chelator. Bacterial growth was evaluated by spectrophotometric OD600 reading. The percent growth was determined by comparing the OD600 reading of each culture to controls grown in medium alone. *p<0.05, **p<0.01, ***p<0.001, Student's t-test, n = 3 biological replicates.
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ppat-1004450-g002: Inhibition of H. pylori growth in vitro by CP or TPEN is dose dependent.A) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 50 µM zinc chloride plus 50 µM manganese chloride (Medium+Zinc+Manganese) and with increasing concentrations of CP. B) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 100 µM zinc chloride (Medium+Zinc) and with increasing concentrations of TPEN, a synthetic zinc chelator. Bacterial growth was evaluated by spectrophotometric OD600 reading. The percent growth was determined by comparing the OD600 reading of each culture to controls grown in medium alone. *p<0.05, **p<0.01, ***p<0.001, Student's t-test, n = 3 biological replicates.

Mentions: CP has been demonstrated to inhibit bacterial growth via sequestration of nutrient manganese and zinc [16]–[18]. We hypothesized that CP is elevated in response to H. pylori infection as part of a host strategy to inhibit bacterial proliferation within the gastric niche. To test this proposal, in vitro growth assays were performed in modified bacteriological medium. Analysis of bacterial growth curves (OD600) and colony forming units (CFU/mL) revealed that wild-type CP (CP) at 300 µg/mL significantly inhibited H. pylori growth (Figure 2 and Figure S1). The addition of exogenous manganese and zinc (50 µM of zinc chloride and 50 µM manganese chloride) restored growth to control levels. In addition to investigating the ability of CP to inhibit growth of H. pylori, the effects of three previously generated mutants of CP's metal binding sites (DS CP, ΔS1, and ΔS2) were investigated [15]. The DS CP harboring inactivation of both S1 (manganese and zinc binding) and S2 (zinc binding alone) sites was unable to inhibit bacterial growth (Table S1). The ΔS1 mutant at 1200 µg/mL was able to inhibit bacterial growth, as was the ΔS2 mutant (Table S1). These results indicate that CP inhibited H. pylori growth in vitro at concentrations above 300 µg/mL, and that the antibacterial activity is dependent on CP's ability to sequester metal.


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)

Inhibition of H. pylori growth in vitro by CP or TPEN is dose dependent.A) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 50 µM zinc chloride plus 50 µM manganese chloride (Medium+Zinc+Manganese) and with increasing concentrations of CP. B) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 100 µM zinc chloride (Medium+Zinc) and with increasing concentrations of TPEN, a synthetic zinc chelator. Bacterial growth was evaluated by spectrophotometric OD600 reading. The percent growth was determined by comparing the OD600 reading of each culture to controls grown in medium alone. *p<0.05, **p<0.01, ***p<0.001, Student's t-test, n = 3 biological replicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199781&req=5

ppat-1004450-g002: Inhibition of H. pylori growth in vitro by CP or TPEN is dose dependent.A) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 50 µM zinc chloride plus 50 µM manganese chloride (Medium+Zinc+Manganese) and with increasing concentrations of CP. B) WT H. pylori were cultured for 24 hours in medium alone or medium supplemented with 100 µM zinc chloride (Medium+Zinc) and with increasing concentrations of TPEN, a synthetic zinc chelator. Bacterial growth was evaluated by spectrophotometric OD600 reading. The percent growth was determined by comparing the OD600 reading of each culture to controls grown in medium alone. *p<0.05, **p<0.01, ***p<0.001, Student's t-test, n = 3 biological replicates.
Mentions: CP has been demonstrated to inhibit bacterial growth via sequestration of nutrient manganese and zinc [16]–[18]. We hypothesized that CP is elevated in response to H. pylori infection as part of a host strategy to inhibit bacterial proliferation within the gastric niche. To test this proposal, in vitro growth assays were performed in modified bacteriological medium. Analysis of bacterial growth curves (OD600) and colony forming units (CFU/mL) revealed that wild-type CP (CP) at 300 µg/mL significantly inhibited H. pylori growth (Figure 2 and Figure S1). The addition of exogenous manganese and zinc (50 µM of zinc chloride and 50 µM manganese chloride) restored growth to control levels. In addition to investigating the ability of CP to inhibit growth of H. pylori, the effects of three previously generated mutants of CP's metal binding sites (DS CP, ΔS1, and ΔS2) were investigated [15]. The DS CP harboring inactivation of both S1 (manganese and zinc binding) and S2 (zinc binding alone) sites was unable to inhibit bacterial growth (Table S1). The ΔS1 mutant at 1200 µg/mL was able to inhibit bacterial growth, as was the ΔS2 mutant (Table S1). These results indicate that CP inhibited H. pylori growth in vitro at concentrations above 300 µg/mL, and that the antibacterial activity is dependent on CP's ability to sequester metal.

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