Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island:: essential genes for CagA translocation in host cells and induction of interleukin-8
Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island:: essential genes for CagA translocation in host cells and induction of interleukin-8
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DOI：
10.1046/j.1365-2958.2001.02714.x
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发表时间：
2001-12-01
影响因子：
3.6
通讯作者：
Haas, R
中科院分区：
文献类型：
--
作者：
Fischer, W;Püls, J;Haas, R
幽门螺杆菌（Hp）携带由cag致病岛（cag-PAI）编码的IV型分泌系统，其用于：（i）将细菌效应蛋白CagA易位到不同类型的真核细胞中;和（ii）诱导趋化因子（如白细胞介素-8（IL-8））的合成和分泌。Hp 26695中的cag-PAI由27个推定基因组成，其中6个被鉴定为根癌农杆菌virB操纵子所代表的基本IV型分泌系统的同源物。为了确定27个基因中每一个的作用和贡献，我们应用了精确的缺失/插入诱变程序来敲除每个单独的基因，而不会对下游基因的表达产生极性效应。发现27个基因中有17个对于CagA易位到宿主细胞中是绝对必需的，并且27个基因中有14个对于Hp完全诱导IL-8转录的能力是绝对必需的。hp0524（virD4同源物）、hp0526和hp0540的产物对于CagA的易位是绝对必需的，但对于IL-8的诱导不是必需的。相反，hp0520、hp0521、hp0534、hp0535、hp0536和hp0543的产物对于CagA的易位或IL-8的诱导不是必需的。我们的数据反对易位IL-8诱导的效应蛋白编码的cag-PAI。我们分离了Hp 26695的变体，其自发地关闭了其IL-8诱导和CagA易位的能力，但保留了完整的cag-PAI。我们确定了基因hp0532的点突变，导致相应的多肽链的翻译提前停止，提供了一个假定的解释IV型分泌系统的自发突变的缺陷。
Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction. Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.