A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.
A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.
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DOI：
10.1371/journal.ppat.1004621
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发表时间：
2015-02
期刊：
影响因子：
6.7
通讯作者：
Blaser MJ
中科院分区：
文献类型：
--
作者：
Zhang XS;Tegtmeyer N;Traube L;Jindal S;Perez-Perez G;Sticht H;Backert S;Blaser MJ
幽门螺杆菌持续定植在人类胃中，在人类健康中扮演着复杂的角色。CagA蛋白是一种关键的宿主相互作用因子，通过IV型分泌系统转运到宿主上皮细胞中，在那里其EPIYA酪氨酸磷酸化基序（TPM）被宿主细胞激酶识别，导致多个宿主细胞信号级联。CagA TPM已被描述为A、B、C或D型，每种都具有围绕EPIYA的特异性保守氨基酸序列。数据库检索显示，B基序（包括EPIYA和EPIYT）在Western H.幽门螺杆菌分离株。Western H. pylori CagA序列提供的证据表明，EPIYT B-TPM与胃癌的相关性显著低于EPIYA B-TPM。通过产生和使用磷酸化的CagA B-TPM特异性抗体，我们证明了在H. pylori与宿主细胞共培养。我们还表明，在宿主细胞内，CagA与磷酸肌醇3-激酶（PI 3-kinase）的相互作用是B-TPM酪氨酸磷酸化依赖性的，与EPIYA B-TPM的重组CagA具有更高的亲和力PI 3-kinase和增强诱导AKT比同基因CagA与EPIYA B-TPM。结合到PI 3-激酶的CagA B-TPM基序的结构建模表明，在pY+1位置的苏氨酸残基形成侧链氢键到PI 3-激酶的N-417，其不能由丙氨酸形成。在与AGS细胞共培养过程中，H.与具有B-TPM EPIYA的同基因菌株相比，具有CagA EPIYT B-TPM的pylori菌株具有显著减弱的白细胞介素-8和蜂鸟表型的诱导。这些结果表明，A/T多态性可能通过干扰与肿瘤发生相关的宿主信号通路，调节CagA活性，从而影响癌症风险。幽门螺杆菌是人类胃内的主要致病菌，对人体健康有着复杂的影响。一个重要的致病风险因素是CagA蛋白，其通过其EPIYA酪氨酸磷酸化基序（TPM）干扰多种宿主细胞信号传导途径。通过数据库检索和计算机模拟分析，我们发现在Western H. pylori分离物，并提供证据表明EPIYT与胃癌的相关性显著低于EPIYA。通过构造一系列H. pylori cagA同基因突变体和同基因互补质粒，产生特异性抗体，与人AGS细胞共培养，进行生物化学和建模分析，我们证明了CagA B基序磷酸化状态对于其在定植期间与宿主PI 3-激酶的相互作用是必需的，并且具有EPIYT B基序的CagA显著减弱了白细胞介素-8和蜂鸟表型的诱导，与EPIYA相比，具有与PI 3-激酶更高的亲和力，并增强AKT的诱导。这些发现提供了深入了解如何西方H。幽门螺杆菌CagA通过功能重要的B基序的A/T多态性调节宿主细胞内的癌症相关活性。
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. 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 B-motif 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.
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影响因子：
4.8
作者：
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通讯作者：
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作者：
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