Jana N. Radin

High Dietary Salt Intake Exacerbates Helicobacter pylori-Induced Gastric Carcinogenesis

ABSTRACT Persistent colonization of the human stomach with Helicobacter pylori is a risk factor for gastric adenocarcinoma, and H. pylori-induced carcinogenesis is dependent on the actions of a bacterial oncoprotein known as CagA. Epidemiological studies have shown that high dietary salt intake is also a risk factor for gastric cancer. To investigate the effects of a high-salt diet, we infected Mongolian gerbils with a wild-type (WT) cagA + H. pylori strain or an isogenic cagA mutant strain and maintained the animals on a regular diet or a high-salt diet. At 4 months postinfection, gastric adenocarcinoma was detected in 100% of the WT-infected/high-salt-diet animals, 58% of WT-infected/regular-diet animals, and none of the animals infected with the cagA mutant strain (P < 0.0001). Among animals infected with the WT strain, those fed a high-salt diet had more severe gastric inflammation, higher gastric pH, increased parietal cell loss, increased gastric expression of interleukin 1β (IL-1β), and decreased gastric expression of hepcidin and hydrogen potassium ATPase (H,K-ATPase) compared to those on a regular diet. Previous studies have detected upregulation of CagA synthesis in response to increased salt concentrations in the bacterial culture medium, and, concordant with the in vitro results, we detected increased cagA transcription in vivo in animals fed a high-salt diet compared to those on a regular diet. Animals infected with the cagA mutant strain had low levels of gastric inflammation and did not develop hypochlorhydria. These results indicate that a high-salt diet potentiates the carcinogenic effects of cagA + H. pylori strains.

Helicobacter pylori VacA Induces Programmed Necrosis in Gastric Epithelial Cells

ABSTRACT Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.

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

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 intermediate region of Helicobacter pylori VacA is a determinant of toxin potency in a Jurkat T cell assay.

ABSTRACT Colonization of the human stomach with Helicobacter pylori is a risk factor for peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. The secreted VacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1, i1, and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2, i2, and m2). Thus far, there has been relatively little study of the role of the VacA intermediate region (i-region) in toxin activity. In this study, we compared the ability of i1 and i2 forms of VacA to cause functional alterations in Jurkat cells. To do this, we manipulated the chromosomal vacA gene in two H. pylori strains to introduce alterations in the region encoding the VacA i-region. We did not detect any differences in the capacity of i1 and i2 forms of VacA to cause vacuolation of RK13 cells. In comparison to i1 forms of VacA, i2 forms of VacA had a diminished capacity to inhibit the activation of nuclear factor of activated T cells (NFAT) and suppress interleukin-2 (IL-2) production. Correspondingly, i2 forms of VacA bound to Jurkat cells less avidly than did i1 forms of VacA. These results indicate that the VacA i-region is an important determinant of VacA effects on human T cell function.

Role of Connexin 43 in Helicobacter pylori VacA-Induced Cell Death

ABSTRACT Helicobacter pylori colonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pylori toxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43 expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.

IL-17a and IL-22 Induce Expression of Antimicrobials in Gastrointestinal Epithelial Cells and May Contribute to Epithelial Cell Defense against Helicobacter pylori

Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in pro-inflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some β-defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17A-activated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.

Flagellar Localization of a Helicobacter pylori Autotransporter Protein

ABSTRACT Helicobacter pylori contains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface of H. pylori. The expression of the three vacA-like genes is upregulated during H. pylori colonization of the mouse stomach compared to H. pylori growth in vitro, and a wild-type H. pylori strain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-type H. pylori strain, an isogenic faaA mutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility. IMPORTANCE The pathogenesis of most bacterial infections is dependent on the actions of secreted proteins, and proteins secreted by the autotransporter pathway constitute the largest family of secreted proteins in pathogenic Gram-negative bacteria. In this study, we analyzed three autotransporter proteins (VacA-like proteins) produced by Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We demonstrate that these three proteins each enhance the capacity of H. pylori to colonize the stomach. Unexpectedly, one of these proteins (FaaA) is localized to a sheath that overlies H. pylori flagella. The absence of FaaA results in decreased H. pylori motility as well as a reduction in flagellar stability and a change in flagellar localization. The atypical localization of FaaA reflects a specialized function of this autotransporter designed to optimize H. pylori colonization of the gastric niche. The pathogenesis of most bacterial infections is dependent on the actions of secreted proteins, and proteins secreted by the autotransporter pathway constitute the largest family of secreted proteins in pathogenic Gram-negative bacteria. In this study, we analyzed three autotransporter proteins (VacA-like proteins) produced by Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We demonstrate that these three proteins each enhance the capacity of H. pylori to colonize the stomach. Unexpectedly, one of these proteins (FaaA) is localized to a sheath that overlies H. pylori flagella. The absence of FaaA results in decreased H. pylori motility as well as a reduction in flagellar stability and a change in flagellar localization. The atypical localization of FaaA reflects a specialized function of this autotransporter designed to optimize H. pylori colonization of the gastric niche.

TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection

Calcium signaling in phagocytes is essential for cellular activation, migration, and the potential resolution of infection or inflammation. The generation of reactive oxygen species (ROS) via activation of NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase activity in macrophages has been linked to altered intracellular calcium concentrations. Because of its role as an oxidative stress sensor in phagocytes, we investigated the function of the cation channel transient receptor potential melastatin 2 (TRPM2) in macrophages during oxidative stress responses induced by Helicobacter pylori infection. We show that Trpm2−/− mice, when chronically infected with H. pylori, exhibit increased gastric inflammation and decreased bacterial colonization compared with wild-type (WT) mice. The absence of TRPM2 triggers greater macrophage production of inflammatory mediators and promotes classically activated macrophage M1 polarization in response to H. pylori. TRPM2-deficient macrophages upon H. pylori stimulation are unable to control intracellular calcium levels, which results in calcium overloading. Furthermore, increased intracellular calcium in TRPM2−/− macrophages enhanced mitogen-activated protein kinase and NADPH-oxidase activities, compared with WT macrophages. Our data suggest that augmented production of ROS and inflammatory cytokines with TRPM2 deletion regulates oxidative stress in macrophages and consequently decreases H. pylori gastric colonization while increasing inflammation in the gastric mucosa.

Helicobacter pylori Resists the Antimicrobial Activity of Calprotectin via Lipid A Modification and Associated Biofilm Formation

ABSTRACT Helicobacter pylori is one of several pathogens that persist within the host despite a robust immune response. H. pylori elicits a proinflammatory response from host epithelia, resulting in the recruitment of immune cells which manifests as gastritis. Relatively little is known about how H. pylori survives antimicrobials, including calprotectin (CP), which is present during the inflammatory response. The data presented here suggest that one way H. pylori survives the nutrient sequestration by CP is through alteration of its outer membrane. CP-treated H. pylori demonstrates increased bacterial fitness in response to further coculture with CP. Moreover, CP-treated H. pylori cultures form biofilms and demonstrate decreased cell surface hydrophobicity. In response to CP, the H. pylori Lpx lipid A biosynthetic enzymes are not fully functional. The lipid A molecules observed in H. pylori cultures treated with CP indicate that the LpxF, LpxL, and LpxR enzyme functions are perturbed. Transcriptional analysis of lpxF, lpxL, and lpxR indicates that metal restriction by CP does not control this pathway through transcriptional regulation. Analyses of H. pylori lpx mutants reveal that loss of LpxF and LpxL results in increased fitness, similar to what is observed in the presence of CP; moreover, these mutants have significantly increased biofilm formation and reduced cell surface hydrophobicity. Taken together, these results demonstrate a novel mechanism of H. pylori resistance to the antimicrobial activity of CP via lipid A modification strategies and resulting biofilm formation. IMPORTANCE Helicobacter pylori evades recognition of the hosts immune system by modifying the lipid A component of lipopolysaccharide. These results demonstrate for the first time that the lipid A modification pathway is influenced by the hosts nutritional immune response. H. pyloris exposure to the host Mn- and Zn-binding protein calprotectin perturbs the function of 3 enzymes involved in the lipid A modification pathway. Moreover, CP treatment of H. pylori, or mutants with an altered lipid A, exhibit increased bacterial fitness and increased biofilm formation. This suggests that H. pylori modifies its cell surface structure to survive under the stress imposed by the host immune response. These results provide new insights into the molecular mechanisms that influence the biofilm lifestyle and how endotoxin modification, which renders H. pylori resistant to cationic antimicrobial peptides, can be inactivated in response to sequestration of nutrient metals. Helicobacter pylori evades recognition of the hosts immune system by modifying the lipid A component of lipopolysaccharide. These results demonstrate for the first time that the lipid A modification pathway is influenced by the hosts nutritional immune response. H. pyloris exposure to the host Mn- and Zn-binding protein calprotectin perturbs the function of 3 enzymes involved in the lipid A modification pathway. Moreover, CP treatment of H. pylori, or mutants with an altered lipid A, exhibit increased bacterial fitness and increased biofilm formation. This suggests that H. pylori modifies its cell surface structure to survive under the stress imposed by the host immune response. These results provide new insights into the molecular mechanisms that influence the biofilm lifestyle and how endotoxin modification, which renders H. pylori resistant to cationic antimicrobial peptides, can be inactivated in response to sequestration of nutrient metals.

The Two-Component System ArlRS and Alterations in Metabolism Enable Staphylococcus aureus to Resist Calprotectin-Induced Manganese Starvation

During infection the host imposes manganese and zinc starvation on invading pathogens. Despite this, Staphylococcus aureus and other successful pathogens remain capable of causing devastating disease. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. We report that ArlRS, a global staphylococcal virulence regulator, enhances the ability of S. aureus to grow in the presence of the manganese-and zinc-binding innate immune effector calprotectin. Utilization of calprotectin variants with altered metal binding properties revealed that strains lacking ArlRS are specifically more sensitive to manganese starvation. Loss of ArlRS did not alter the expression of manganese importers or prevent S. aureus from acquiring metals. It did, however, alter staphylococcal metabolism and impair the ability of S. aureus to grow on amino acids. Further studies suggested that relative to consuming glucose, the preferred carbon source of S. aureus, utilizing amino acids reduced the cellular demand for manganese. When forced to use glucose as the sole carbon source S. aureus became more sensitive to calprotectin compared to when amino acids are provided. Infection experiments utilizing wild type and calprotectin-deficient mice, which have defects in manganese sequestration, revealed that ArlRS is important for disease when manganese availability is restricted but not when this essential nutrient is freely available. In total, these results indicate that altering cellular metabolism contributes to the ability of pathogens to resist manganese starvation and that ArlRS enables S. aureus to overcome nutritional immunity by facilitating this adaptation.