The Host Protein Calprotectin Modulates the Helicobacter pylori cag Type IV Secretion System via Zinc Sequestration

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.

The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence

“…For coculture assays, bacteria were subcultured in 60% brucella broth plus 40% calprotectin buffer supplemented with cholesterol, referred to as "medium alone" (27), or medium supplemented with 500 g/ml (23.9 M) purified S100A12 protein alone or with 100 M zinc chloride or 100 M copper chloride. For zinc titration experiments, bacteria were grown in the presence of 500 g/ml (23.9 M) purified S100A12 protein alone or supplemented with 1.25 M, 2.5 M, 5 M, 10 M, or 25 M zinc chloride.…”

“…Five images per case and antibody were obtained in areas of the mucosa with the highest concentration of positive cells. Micrograph images were analyzed with the ImageJ IHC toolbox plugin to quantify H-DAB staining by color detection and converted to 8-bit format, and densitometry quantification was carried out as previously described (27).…”

“…Secreted IL-8 was evaluated by Quantikine human IL-8 sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems) per the manufacturer's instructions. IL-8 values were compared to those derived from coculture samples from WT bacteria grown in medium alone as previously described (27).…”

“…Bacterial adherence was evaluated as previously described (27). Briefly, cocultures of 10 5 AGS gastric epithelial cells and H. pylori (MOI of 100:1) were performed as described above, and cells were scraped into 1 ml of sterile phosphate-buffered saline (pH 7.4), serially diluted, and plated onto blood agar plates.…”

“…Numerous pathogens, including Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Escherichia coli, Borrelia burgdorferi, Listeria monocytogenes, Candida albicans, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococcus faecalis, Pseudomonas aeruginosa, and Shigella flexneri, have been shown to be susceptible to the antimicrobial activity of the S100A8/S100A9 heterodimer calprotectin (19)(20)(21)(22)(23)(24)(25)(26). Our recent work demonstrated that calprotectin represses the growth and virulence of H. pylori by sequestering nutrient zinc (27). However, the contribution of additional S100 proteins to growth and virulence repression in H. pylori remains unknown.…”