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.
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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)