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

Abstract

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.

Differences in amino acid frequency in CagA and VacA sequences of Helicobacter pylori distinguish gastric cancer from gastric MALT lymphoma

“…Tyrosine phosphorylation of EPIYA/T B-TPMs were reported to be involved in the regulation of host cell signaling cascades and interacted with epithelial cells [30]. In this study, most strains possessed EPIYA, not EPIYT B-TPMs, which were in agreement with the previous report that most East Asian strains (91.1%) possessed EPIYA B-TPMs [30].

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“…Recent data revealed a strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in CagAs. The EPIYT B-tyrosine phosphorylation motifs (TPMs) were significantly less associated with GC than the EPIYA B-TPMs [30]. In this study, most strains possessed EPIYA B-TPMs, except for three strains with ESIYA or ESIYT B-TPMs (one strain from GC had ESIYA B-TPM, and one strain from gastritis and one strain from MALT lymphoma had ESIYT B-TPM).…”

Pathobiology of Helicobacter pylori–Induced Gastric Cancer

“…The number and types of repeats have been associated with differing risks of carcinogenesis 58 . Recently, it was reported that the EPIYA-B motif binds and activates PI3-kinase and AKT, but many Western strains of H pylori carry a single nucleotide polymorphism in this domain that reduces its association with PI3 kinase and also reduces the risk of gastric cancer 59 . The amount of CagA and its cellular target are also likely to have important roles in pathogenesis, but little is known about the precise location and timing CagA delivery.…”

Helicobacter pylorivirulence genes and host genetic polymorphisms as risk factors for peptic ulcer disease

“…The type and number of CM motifs may influence the potential for individual CagA proteins to multimerize in host cells, and this may affect the ability of CagA to disturb host cell function via SHP-2 deregulation [33]. The EPIYT is the second most common EPIYA-B motif in Western-type cagA , but is very rarely observed in East-Asian-type- cagA [34]. Zhang et al .…”

“…Zhang et al . analyzed 364 Western-type- cagA and reported that gastric cancer is more significantly associated with EPIYA sequences in the EPIYA-B motif compared with gastritis alone, whereas EPIYT sequences are significantly associated with DU [34]. …”