The repetitive domain of ScARP3d triggers entry of Spiroplasma citri into cultured cells of the vector Circulifer haematoceps.
The repetitive domain of ScARP3d triggers entry of Spiroplasma citri into cultured cells of the vector Circulifer haematoceps.
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ScARP3d 的重复结构域触发柑橘螺原体进入载体Circulifer haematoceps 的培养细胞中。
DOI：
10.1371/journal.pone.0048606
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Arricau-Bouvery N
中科院分区：
文献类型：
--
作者：
Béven L;Duret S;Batailler B;Dubrana MP;Saillard C;Renaudin J;Arricau-Bouvery N
柑橘螺原体是一种植物病原性软体动物，由叶蝉载体Circulifer haematoceps 传播。成功的传播需要螺原体通过受体-配体相互作用介导的内吞作用穿过肠上皮和唾液腺屏障。为了表征这些相互作用，我们使用 Ciha-1 叶蝉细胞系研究了 S. citri 突变体的粘附和入侵能力。 S. citri GII3 野生型包含 7 个质粒，其中 5 个（pSci1 至 5）编码 8 个相关粘附素 (ScARP)。与野生型菌株GII3相比，缺乏pSci1至5的柑橘链霉菌突变体G/6其粘附和进入Ciha-1细胞的能力受到影响。蛋白水解分析、Triton X-114 分配和凝集测定表明，ScARP3d 的 N 端部分（由重复序列组成）暴露于螺原体表面，而 C 端部分则锚定在膜中。乳胶珠细胞粘附测定显示 ScARP3d 重复结构域 (Rep3d) 参与其中，并且 Rep3d 包被的珠子的内化是肌动蛋白依赖性的。这些数据表明 ScARP3d 通过其 Rep3d 结构域与 S. citri GII3 与昆虫细胞的粘附有关。使用抗 Rep3d 抗体的抑制测试和重组 Rep3d 的竞争性测定均导致螺原体对昆虫细胞入侵的减少。出乎意料的是，用肌动蛋白聚合抑制剂细胞松弛素 D 处理 Ciha-1 细胞增加了粘附，从而增加了 S. citri GII3 的进入。对于无 ScARPs 的突变体 G/6，在细胞松弛素 D 处理后仅粘附增强，尽管程度较小。总之，这些结果强烈表明 ScARP，特别是 ScARP3d，在柑橘链霉菌对叶蝉细胞的粘附和侵袭中的作用。
Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.
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