A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

PLoS Pathogens, Feb 2015

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

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A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

February A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions Xue-Song Zhang 0 1 Nicole Tegtmeyer 0 1 Leah Traube 0 1 Shawn Jindal 0 1 Guillermo Perez- Perez 0 1 Heinrich Sticht 0 1 Steffen Backert 0 1 Martin J. Blaser 0 1 0 1 Departments of Medicine and Microbiology, New York University School of Medicine and VA Medical Center , New York , New York, United States of America, 2 Friedrich Alexander University Erlangen, Department of Biology, Division of Microbiology, Erlangen, Germany, 3 Friedrich Alexander University Erlangen, Bioinformatics, Institute for Biochemistry , Erlangen , Germany 1 Editor: Steven R. Blanke, University of Illinois, UNITED STATES Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During coculture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk. - Funding: The work of XSZ, LT, SJ, GPP, and MJB is supported by the National Institutes of Health (R01GM63270), by the Knapp Family Fund, the Ziff Foundation, and the Diane Belfer Program for Human Microbial Ecology. The work of NT, SB, and HS is supported through DFG grants (projects B10 and A2 of CRC-796). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. As the dominant bacterium living in the human stomach, Helicobacter pylori has mixed roles in host health. One significant pathogenic risk factor is the CagA protein, which interferes with multiple host cell signaling pathways through its EPIYA tyrosine phosphorylation motifs (TPMs). Through database searching and silico analysis, we reveal a strong non-random distribution of the EPIYA B motif polymorphisms (including EPIYT and EPIYA) in Western H. pylori isolates, and provide evidence that the EPIYT are significantly less associated with gastric cancer than the EPIYA. By constructing a series of H. pylori cagA isogenic mutants and isogenic complementation plasmids, generating specific antibodies, co-culturing with human AGS cells, performing biochemical and modeling analysis, we demonstrate that CagA B-motif phosphorylation status is essential for its interaction with host PI3-kinase during colonization and that CagA with an EPIYT Bmotif had significantly attenuated induction of interleukin-8 and the hummingbird phenotype, had higher affinity with PI3-kinase, and enhanced induction of AKT compared to the EPIYA. These findings provide insight into how Western H. pylori CagA regulates cancer-related activity inside host cells through the A/T polymorphisms at the functionally important B motif. Helicobacter pylori, a spiral-shaped, microaerophilic gram-negative bacterium, persistently colonizes the human gastric mucosa [1,2]. H. pylori is carried by about 50% of the worlds population, and it exhibits extensive genetic diversity and distinct phylogeographic features [3,4]. Colonization increases risk of peptic ulcer disease and gastric carcinoma [5,6], and has been associated with diminished risk for esophageal inflammatory and neoplastic lesions [7,8], and childhood-onset asthma [9,10]. In 1995, the cytotoxin-associated gene (...truncated)


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Xue-Song Zhang, Nicole Tegtmeyer, Leah Traube, Shawn Jindal, Guillermo Perez-Perez, Heinrich Sticht, Steffen Backert, Martin J. Blaser. A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions, PLoS Pathogens, 2015, 2, DOI: 10.1371/journal.ppat.1004621