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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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Host CP (S100A8/A9) is elevated in H. pylori infected stomach tissue.A) s100a8/s100a9 transcript abundance in RNA extracted from C57BL/6 mice infected with H. pylori PMSS1 or SS1 for 1, 2, or 3 months relative to uninfected animals as determined by real-time RT-PCR analysis. Points indicate mean relative units of transcript abundance +/− SEM (levels of s100a8 in PMSS1-infected mice compared to uninfected mice; 1 mo p = 0.0511; 2 mo p = 0.0432; 3 mo p = 0.0127; levels of s100a8 in SS1-infected mice compared to uninfected mice at 2 mo p = 0.0623 Student's t test). (B) Inflammation scores of H. pylori infected mice at 1, 2, and 3 months post infection. (C) s100a8/s100a9 transcript abundance in RNA extracted from gastric biopsies derived from human patients, which were either H. pylori-positive or H. pylori-negative (s100a8 p = 0.15; s100a9 *p = 0.05). Bars indicate mean relative units of transcript abundance +/− SEM. Each experimental group represents≥5 individuals (mice or human samples). D) Gastric samples derived from H. pylori PMSS1-infected WT mice or SS1-infected WT mice at 2 months post-infection were analyzed via immunohistochemistry using a polyclonal antibody to S100A9 (scale bars are 50 microns). E) Real-time RT-PCR was performed on gastric tissue to quantify s100a8 and s100a9 transcript abundance from WT (C57BL/6 mice) and IL-17RA-/- mice infected with PMSS1. Data represent relative units of transcript abundance +/− SEM in WT mice and IL-17RA-/- mice, *p = 0.0169 and p = 0.0143, respectively.
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ppat-1004450-g001: Host CP (S100A8/A9) is elevated in H. pylori infected stomach tissue.A) s100a8/s100a9 transcript abundance in RNA extracted from C57BL/6 mice infected with H. pylori PMSS1 or SS1 for 1, 2, or 3 months relative to uninfected animals as determined by real-time RT-PCR analysis. Points indicate mean relative units of transcript abundance +/− SEM (levels of s100a8 in PMSS1-infected mice compared to uninfected mice; 1 mo p = 0.0511; 2 mo p = 0.0432; 3 mo p = 0.0127; levels of s100a8 in SS1-infected mice compared to uninfected mice at 2 mo p = 0.0623 Student's t test). (B) Inflammation scores of H. pylori infected mice at 1, 2, and 3 months post infection. (C) s100a8/s100a9 transcript abundance in RNA extracted from gastric biopsies derived from human patients, which were either H. pylori-positive or H. pylori-negative (s100a8 p = 0.15; s100a9 *p = 0.05). Bars indicate mean relative units of transcript abundance +/− SEM. Each experimental group represents≥5 individuals (mice or human samples). D) Gastric samples derived from H. pylori PMSS1-infected WT mice or SS1-infected WT mice at 2 months post-infection were analyzed via immunohistochemistry using a polyclonal antibody to S100A9 (scale bars are 50 microns). E) Real-time RT-PCR was performed on gastric tissue to quantify s100a8 and s100a9 transcript abundance from WT (C57BL/6 mice) and IL-17RA-/- mice infected with PMSS1. Data represent relative units of transcript abundance +/− SEM in WT mice and IL-17RA-/- mice, *p = 0.0169 and p = 0.0143, respectively.

Mentions: In order to determine if CP is elevated in the context of H. pylori infection, real-time RT-PCR analysis of s100a8 and s100a9 transcripts in RNA isolated from either mouse or human gastric tissue was performed. For the first mouse study, RNA was isolated from gastric tissue of mice that had been infected with H. pylori PMSS1 or SS1 for 1, 2, and 3 months and s100a8 and s100a9 expression was compared to that of uninfected mice. CP subunit s100a8 was significantly increased in gastric tissue in response to H. pylori infection (Figure 1A). Transcript levels of CP subunit s100a9 did not significantly increase, but it has been demonstrated that the S100A9 protein is stabilized by increased S100A8 expression [18]. Corresponding inflammation scores are presented for these infections (Figure 1B). In a second study, human gastric biopsy samples were divided into H. pylori-negative samples and H. pylori-positive samples. Gene expression of both s100a8 and s100a9 subunits were elevated in H. pylori-infected biopsy samples compared to H. pylori-negative samples (Figure 1C).


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)

Host CP (S100A8/A9) is elevated in H. pylori infected stomach tissue.A) s100a8/s100a9 transcript abundance in RNA extracted from C57BL/6 mice infected with H. pylori PMSS1 or SS1 for 1, 2, or 3 months relative to uninfected animals as determined by real-time RT-PCR analysis. Points indicate mean relative units of transcript abundance +/− SEM (levels of s100a8 in PMSS1-infected mice compared to uninfected mice; 1 mo p = 0.0511; 2 mo p = 0.0432; 3 mo p = 0.0127; levels of s100a8 in SS1-infected mice compared to uninfected mice at 2 mo p = 0.0623 Student's t test). (B) Inflammation scores of H. pylori infected mice at 1, 2, and 3 months post infection. (C) s100a8/s100a9 transcript abundance in RNA extracted from gastric biopsies derived from human patients, which were either H. pylori-positive or H. pylori-negative (s100a8 p = 0.15; s100a9 *p = 0.05). Bars indicate mean relative units of transcript abundance +/− SEM. Each experimental group represents≥5 individuals (mice or human samples). D) Gastric samples derived from H. pylori PMSS1-infected WT mice or SS1-infected WT mice at 2 months post-infection were analyzed via immunohistochemistry using a polyclonal antibody to S100A9 (scale bars are 50 microns). E) Real-time RT-PCR was performed on gastric tissue to quantify s100a8 and s100a9 transcript abundance from WT (C57BL/6 mice) and IL-17RA-/- mice infected with PMSS1. Data represent relative units of transcript abundance +/− SEM in WT mice and IL-17RA-/- mice, *p = 0.0169 and p = 0.0143, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199781&req=5

ppat-1004450-g001: Host CP (S100A8/A9) is elevated in H. pylori infected stomach tissue.A) s100a8/s100a9 transcript abundance in RNA extracted from C57BL/6 mice infected with H. pylori PMSS1 or SS1 for 1, 2, or 3 months relative to uninfected animals as determined by real-time RT-PCR analysis. Points indicate mean relative units of transcript abundance +/− SEM (levels of s100a8 in PMSS1-infected mice compared to uninfected mice; 1 mo p = 0.0511; 2 mo p = 0.0432; 3 mo p = 0.0127; levels of s100a8 in SS1-infected mice compared to uninfected mice at 2 mo p = 0.0623 Student's t test). (B) Inflammation scores of H. pylori infected mice at 1, 2, and 3 months post infection. (C) s100a8/s100a9 transcript abundance in RNA extracted from gastric biopsies derived from human patients, which were either H. pylori-positive or H. pylori-negative (s100a8 p = 0.15; s100a9 *p = 0.05). Bars indicate mean relative units of transcript abundance +/− SEM. Each experimental group represents≥5 individuals (mice or human samples). D) Gastric samples derived from H. pylori PMSS1-infected WT mice or SS1-infected WT mice at 2 months post-infection were analyzed via immunohistochemistry using a polyclonal antibody to S100A9 (scale bars are 50 microns). E) Real-time RT-PCR was performed on gastric tissue to quantify s100a8 and s100a9 transcript abundance from WT (C57BL/6 mice) and IL-17RA-/- mice infected with PMSS1. Data represent relative units of transcript abundance +/− SEM in WT mice and IL-17RA-/- mice, *p = 0.0169 and p = 0.0143, respectively.
Mentions: In order to determine if CP is elevated in the context of H. pylori infection, real-time RT-PCR analysis of s100a8 and s100a9 transcripts in RNA isolated from either mouse or human gastric tissue was performed. For the first mouse study, RNA was isolated from gastric tissue of mice that had been infected with H. pylori PMSS1 or SS1 for 1, 2, and 3 months and s100a8 and s100a9 expression was compared to that of uninfected mice. CP subunit s100a8 was significantly increased in gastric tissue in response to H. pylori infection (Figure 1A). Transcript levels of CP subunit s100a9 did not significantly increase, but it has been demonstrated that the S100A9 protein is stabilized by increased S100A8 expression [18]. Corresponding inflammation scores are presented for these infections (Figure 1B). In a second study, human gastric biopsy samples were divided into H. pylori-negative samples and H. pylori-positive samples. Gene expression of both s100a8 and s100a9 subunits were elevated in H. pylori-infected biopsy samples compared to H. pylori-negative samples (Figure 1C).

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