Mark S. McClain

The Helicobacter pylori Cag Type IV Secretion System (T4SS) and the secreted CagA effector protein have key roles in gastric cancer pathogenesis. The Cag T4SS outer membrane core complex (OMCC) consists of an outer membrane cap (OMC), a periplasmic ring (PR), and a stalk. CagX and CagY span both the OMC (14 copies) and PR (17 copies) subassemblies of the OMCC. CagX is structurally related to VirB9 proteins found in other bacterial T4SSs, except for a unique 130-amino-acid region, designated as the periplasmic insertion (PI) domain. In this study, we investigated the contributions of individual CagX domains to Cag T4SS activity and OMCC assembly by engineering H. pylori strains that produce only the CagX OMC domain, only the CagX PR domain, the CagX OMC and PR domains produced as independent proteins (Split CagX), or a CagX protein lacking the PI domain (CagX Δ131–260). We found that the OMC, PR, and PI domains of CagX are each essential for Cag T4SS activity. Electron microscopy analyses revealed that the Split CagX mutant could assemble the Cag T4SS PR (17 copies of CagX and CagY), and the CagX Δ131–260 mutant could assemble partially intact OMCCs. Crosslinking mass spectrometry analysis showed that the CagX Δ131–260 protein retained spatial relationships with the OMCC components CagY and CagM. An intact CagX protein was required for the association of CagW, CagH, CagI, and CagL with the OMCC. These results provide novel insights into the contributions of individual CagX domains to the assembly and activity of the Cag T4SS.

TonB-dependent transport systems (consisting of TonB-dependent transporters, TonB, ExbB, and ExbD) facilitate uptake of nutrients across the bacterial outer membrane. Tol-Pal systems contain components related to those of TonB systems. In this study, we investigated TonB-dependent transport systems in Helicobacter pylori. H. pylori genomes contain three tonB paralogs (designated tonB1 , tonB2 , and tonB3/tolA ), three exbB paralogs, three exbD paralogs, six genes encoding putative TonB-dependent transporters, tolB , and pal . By using immunopurification and mass spectrometry methods, we show that TonB2, ExbB2, and ExbD2 co-purify with each other, consistent with the corresponding genes being in the same predicted operon. The tonB1 gene is not linked to exbB or exbD genes, but we show that ExbB3 and ExbD3 co-purify with TonB1. The tonB3/tolA gene is in a predicted operon containing exbB1/tolQ , exbD1/tolR , tolB , and pal . We show that TonB3/TolA co-purifies with ExbB1/TolQ and ExbD1/TolR, suggesting that TonB3/TolA is a component of the Tol-Pal system. We show that H. pylori tonB1 is essential for H. pylori growth, tonB2 mutants require supplemental iron for growth, and tonB3 / tolA is non-essential for growth in vitro . ICP-MS experiments showed that a tonB2 mutant contains reduced levels of nickel compared to a wild-type strain, and correspondingly, the tonB2 mutant exhibits reduced urease activity. These results indicate that the TonB2 system has an important role in H. pylori acquisition of nickel and iron. We propose that the TonB1 system has a key role in the acquisition of multiple nutrients required for H. pylori viability.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) deliver an effector protein (CagA) and non-protein substrates into gastric cells through a process that requires the Cag type IV secretion system (T4SS). The Cag T4SS outer membrane core complex (OMCC) contains multiple copies of five proteins, two of which are species-specific proteins. By using modifications of a previously described OMCC immunopurification method and optimized mass spectrometric methods, we have now isolated additional cag PAI-encoded proteins that are present in lower relative abundance. Four of these proteins (CagW, CagL, CagI, and CagH) do not exhibit sequence relatedness to T4SS components in other bacterial species. Size exclusion chromatography analysis of immunopurified samples revealed that CagW, CagL, CagI, and CagH co-elute with OMCC components. These four Cag proteins are copurified with the OMCC in immunopurifications from a Δ cag3 mutant strain (lacking peripheral OMCC components), but not from a Δ cagX mutant strain (defective in OMCC assembly). Negative stain electron microscopy analysis indicated that OMCC preparations isolated from Δ cagW, cagL::kan, Δ cagI, and Δ cagH mutant strains are indistinguishable from wild-type OMCCs. In summary, by using several complementary methods, we have identified multiple species-specific Cag proteins that are associated with the Cag T4SS OMCC and are required for T4SS activity.

Colonization of the human stomach with cag pathogenicity island (PAI)-positive Helicobacter pylori strains is associated with increased gastric cancer risk compared to colonization with cag PAI-negative strains. To evaluate the contributions of the Cag type IV secretion system (T4SS) and CagA (a secreted bacterial oncoprotein) to gastric molecular alterations relevant for carcinogenesis, we infected Mongolian gerbils with a Cag T4SS-positive wild-type (WT) H. pylori strain, one of two Cag T4SS mutant strains (∆ cagT or ∆ cagY ), or a ∆ cagA mutant for 12 weeks. Histologic staining revealed a biphasic distribution of gastric inflammation severity in WT-infected animals and minimal inflammation in animals infected with mutant strains. Atrophic gastritis (a premalignant condition), dysplasia, and gastric adenocarcinoma were only detected in WT-infected animals with high inflammation scores. Transcriptional profiling, liquid chromatography-tandem mass spectrometry analysis of micro-extracted tryptic peptides, and imaging mass spectrometry revealed more than a thousand molecular alterations in gastric tissues from WT-infected animals with high inflammation scores compared to uninfected tissues and few alterations in tissues from other groups of infected animals. Proteins with altered abundance in animals with severe Cag T4SS-induced inflammation mapped to multiple pathways, including the complement/coagulation cascade and proteasome pathway. Proteins exhibiting markedly increased abundance in tissues from H. pylori -infected animals with severe inflammation included calprotectin components, proteins involved in proteasome activation, polymeric immunoglobulin receptor (PIGR), interferon-inducible guanylate-binding protein (GBP2), lactoferrin, lysozyme, superoxide dismutase, and eosinophil peroxidase. These results demonstrate key roles for CagA and Cag T4SS activity in promoting gastric mucosal inflammation, transcriptional alterations, and proteomic alterations relevant to gastric carcinogenesis. IMPORTANCE: Helicobacter pylori colonizes the stomachs of about half of humans worldwide, and its presence is the primary risk factor for the development of stomach cancer. H. pylori strains isolated from humans can be broadly classified into two groups based on whether they contain a chromosomal cag pathogenicity island, which encodes a secreted effector protein (CagA) and components of a type IV secretion system (T4SS). In experiments using a Mongolian gerbil model, we found that severe gastric inflammation and gastric transcriptional and proteomic alterations related to gastric cancer development were detected only in animals infected with a wild-type H. pylori strain containing CagA and an intact Cag T4SS . Mutant strains lacking CagA or Cag T4SS activity successfully colonized the stomach without inducing detectable pathologic host responses. These findings illustrate two different patterns of H. pylori -host interaction.

Helicobacter pylori infection is the predominant risk factor for the development of gastric cancer. Risk is enhanced by specific H. pylori virulence factors, diet, and the inflammatory response. Chronic activation of T helper (Th) 1 and Th17 pathways contributes to prolonged inflammation; yet, higher expression of IL-17 receptor (IL-17RA) is a favorable prognostic marker for survival after gastric cancer diagnosis. The protective impact of IL-17RA signaling is not understood. To investigate if IL-17RA signaling protects during H. pylori-induced carcinogenesis, the transgenic InsGAS^tg/tg mouse, which is prone to H. pylori-induced gastric cancer, was utilized. InsGAS^tg/tg mice and InsGAS^tg/tgIl17ra^-/- mice were infected with a cag type 4 secretion system (T4SS) positive H. pylori strain for up to 6 months. Six weeks post-infection, IL-17RA deficiency led to increased bacterial burden, increased gastritis, and development of lymphoid follicles. Increased inflammation was associated with heightened cellular proliferation and earlier loss of parietal and chief cells in InsGAS^tg/tgIl17ra^-/- mice. Gastric cancers developed more frequently by 3- and 6-months post-infection in H. pylori-infected InsGAS^tg/tgIl17ra^-/- mice compared to InsGAS^tg/tg mice. Chronic inflammation was exacerbated with IL-17RA deficiency, characterized by elevated Th1/Th17 cytokines, increased B cell infiltration, and enhanced IgA production, despite reduced expression of the polymeric immunoglobulin receptor. Further, paragastric lymph nodes of InsGAS^tg/tgIl17ra^-/- mice were enlarged relative to controls and displayed altered gene expression profiles. Increased inflammation was accompanied by a significant increase in Cybb expression, which encodes NADPH oxidase 2, suggesting that increased oxidative damage may occur in the absence of IL-17RA. Further, there is increased phosphorylation of histone 2AX in IL-17RA deficient mice, indicating that the DNA damage response is highly activated. These data suggest that IL-17RA signaling activates a protective pathway to prevent excessive inflammation which otherwise can lead to increased oxidative stress, DNA damage, and drive gastric carcinogenesis after H. pylori infection.

Colonization of the human stomach with Helicobacter pylori strains producing active forms of the secreted toxin VacA is associated with an increased risk of peptic ulcer disease and gastric cancer, compared with colonization with strains producing hypoactive forms of VacA. Previous studies have shown that active s1m1 forms of VacA cause cell vacuolation and mitochondrial dysfunction. In this study, we sought to define the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic analyses revealed that several hundred metabolites were significantly altered in VacA-treated gastroduodenal cells (AGS and AZ-521) compared with control cells. Pathway analysis suggested that VacA caused alterations in taurine and hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of VacA, but not hypoactive s2m1 or Δ6–27 VacA-mutant proteins (defective in membrane channel formation), caused reductions in intracellular taurine and hypotaurine concentrations. Supplementation of the tissue culture medium with taurine or hypotaurine protected AZ-521 cells against VacA-induced cell death. Untargeted global metabolomics of VacA-treated AZ-521 cells or AGS cells in the presence or absence of extracellular taurine showed that taurine was the main intracellular metabolite significantly altered by extracellular taurine supplementation. These results indicate that VacA causes alterations in cellular taurine metabolism and that repletion of taurine is sufficient to attenuate VacA-induced cell death. We discuss these results in the context of previous literature showing the important role of taurine in cell physiology and the pathophysiology or treatment of multiple pathologic conditions, including gastric ulcers, cardiovascular disease, malignancy, inflammatory diseases, and other aging-related disorders.

Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. Helicobacter pylori lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems. The number and variety of acyl chains on lipoproteins impact the innate immune response through Toll-like receptor 2. The acyl chains added to lipoproteins are derived from membrane phospholipids. H. pylori membrane phospholipids have previously been shown to consist primarily of C14:0 and C19:0 cyclopropane-containing acyl chains. However, the acyl composition of H. pylori lipoproteins has not been determined. In this study, we characterized the acyl composition of two representative H. pylori lipoproteins, Lpp20 and CagT. Fatty acid methyl esters were prepared from both purified lipoproteins and analyzed by gas chromatography-mass spectrometry. For comparison, we also analyzed H. pylori phospholipids. Consistent with previous studies, we observed that the H. pylori phospholipids contain primarily C14:0 and C19:0 cyclopropane-containing fatty acids. In contrast, both the ester-linked and amide-linked fatty acids found in H. pylori lipoproteins were observed to be almost exclusively C16:0 and C18:0. A discrepancy between the acyl composition of membrane phospholipids and lipoproteins as reported here for H. pylori has been previously reported in other bacteria including Borrelia and Brucella . We discuss possible mechanisms. IMPORTANCE: Colonization of the stomach by Helicobacter pylori is an important risk factor in the development of gastric cancer, the third leading cause of cancer-related death worldwide. H. pylori persists in the stomach despite an immune response against the bacteria. Recognition of lipoproteins by TLR2 contributes to the innate immune response to H. pylori . However, the role of H. pylori lipoproteins in bacterial persistence is poorly understood. As the host response to lipoproteins depends on the acyl chain content, defining the acyl composition of H. pylori lipoproteins is an important step in characterizing how lipoproteins contribute to persistence.

Helicobacter pylori colonization of the human stomach is a strong risk factor for gastric cancer. To investigate H. pylori -induced gastric molecular alterations, we used a Mongolian gerbil model of gastric carcinogenesis. Histologic evaluation revealed varying levels of atrophic gastritis (a premalignant condition characterized by parietal and chief cell loss) in H. pylori -infected animals, and transcriptional profiling revealed a loss of markers for these cell types. We then assessed the spatial distribution and relative abundance of proteins in the gastric tissues using imaging mass spectrometry and liquid chromatography with tandem mass spectrometry. We detected striking differences in the protein content of corpus and antrum tissues. Four hundred ninety-two proteins were preferentially localized to the corpus in uninfected animals. The abundance of 91 of these proteins was reduced in H. pylori -infected corpus tissues exhibiting atrophic gastritis compared with infected corpus tissues exhibiting non-atrophic gastritis or uninfected corpus tissues; these included numerous proteins with metabolic functions. Fifty proteins localized to the corpus in uninfected animals were diffusely delocalized throughout the stomach in infected tissues with atrophic gastritis; these included numerous proteins with roles in protein processing. The corresponding alterations were not detected in animals infected with a H. pylori ∆ cagT mutant (lacking Cag type IV secretion system activity). These results indicate that H. pylori can cause loss of proteins normally localized to the gastric corpus as well as diffuse delocalization of corpus-specific proteins, resulting in marked changes in the normal gastric molecular partitioning into distinct corpus and antrum regions. IMPORTANCE: A normal stomach is organized into distinct regions known as the corpus and antrum, which have different functions, cell types, and gland architectures. Previous studies have primarily used histologic methods to differentiate these regions and detect H. pylori -induced alterations leading to stomach cancer. In this study, we investigated H. pylori -induced gastric molecular alterations in a Mongolian gerbil model of carcinogenesis. We report the detection of numerous proteins that are preferentially localized to the gastric corpus but not the antrum in a normal stomach. We show that stomachs with H. pylori- induced atrophic gastritis (a precancerous condition characterized by the loss of specialized cell types) exhibit marked changes in the abundance and localization of proteins normally localized to the gastric corpus. These results provide new insights into H. pylori -induced gastric molecular alterations that are associated with the development of stomach cancer.

Bacterial lipoproteins are post-translationally modified with acyl chains, anchoring these proteins to bacterial membranes. In Gram-negative bacteria, three enzymes complete the modifications. Lgt (which adds two acyl chains) and LspA (which removes the signal peptide) are essential. Lnt (which adds a third acyl chain) is not essential in certain bacteria including Francisella tularensis , Neisseria gonorrhoeae , and Acinetobacter baumannii . Deleting lnt results in mild to severe physiologic changes. We previously showed lnt is not essential for Helicobacter pylori growth in vitro . Here, the physiologic consequences of deleting lnt in H. pylori and the role of Lnt in the host response to H. pylori were examined using in vitro and in vivo models. Comparing wild-type, Δ lnt , and complemented mutant H. pylori , no changes in growth rates or sensitivity to acid or antibiotics were observed. Since deleting lnt changes the number of acyl chains on lipoproteins and the number of acyl chains on lipoproteins impacts the innate immune response through Toll-like receptor 2 (TLR2) signaling, primary human gastric epithelial cells were treated with a purified lipoprotein from wild-type or lnt mutant H. pylori . Differential gene expression analysis indicated that lipoprotein from the lnt mutant induced a more robust TLR2 response. In a complementary approach, we infected wild-type and Tlr2 ^−/− mice and found that both the wild-type and complemented mutant strains successfully colonized the animals. However, the lnt mutant strain was unable to colonize either mouse strain. These results show that lnt is essential for H. pylori colonization and identifies lipoprotein synthesis as a target for therapeutic intervention.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) are associated with the development of gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a secreted effector protein (CagA) and a type IV secretion system (Cag T4SS). Cag T4SS activity is required for the delivery of CagA and non-protein substrates into host cells. The Cag T4SS outer membrane core complex (OMCC) contains a channel-like domain formed by helix-loop-helix elements (antenna projections, AP) from 14 copies of the CagY protein (a VirB10 ortholog). Similar VirB10 antenna regions are present in T4SS OMCCs from multiple bacterial species and are predicted to span the outer membrane. In this study, we investigated the role of the CagY antenna region in Cag T4SS OMCC assembly and Cag T4SS function. An H. pylori mutant strain with deletion of the entire CagY AP (∆AP) retained the capacity to produce CagY and assemble an OMCC, but it lacked T4SS activity (CagA translocation and IL-8 induction in AGS gastric epithelial cells). In contrast, a mutant strain with Gly-Ser substitutions in the unstructured CagY AP loop retained Cag T4SS activity. Mutants containing CagY AP loops with shortened lengths were defective in CagA translocation and exhibited reduced IL-8-inducing activity compared to control strains. These data indicate that the CagY AP region is required for Cag T4SS activity and that Cag T4SS activity can be modulated by altering the length of the CagY AP unstructured loop.