Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion
Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion
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DOI：
10.1128/iai.00841-19
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发表时间：
2020-05-01
影响因子：
3.1
通讯作者：
Jewett, Travis J.
中科院分区：
文献类型：
--
作者：
Ghosh, Susmita;Ruelke, Elizabeth A.;Jewett, Travis J.
易位肌动蛋白募集磷酸化蛋白（Tarp）是沙眼衣原体使用的多结构域III型分泌效应物。总的来说，现有的数据表明这种效应在引发新感染方面的作用。随着研究衣原体体内基因的新遗传工具的出现，我们推测了Tarp功能在多大程度上促进了衣原体寄生于哺乳动物宿主细胞的能力。为了解决这个问题，我们使用荧光报告的等位基因交换突变（FRAEM）技术生成了一个完整的tarP缺失突变体，并在反式突变体中添加野生型tarP或突变型tarP等位基因，以包含帧内结构域缺失。我们为Tarp在沙眼衣原体侵袭宿主细胞中的重要作用提供了证据。互补研究表明，c端丝状肌动蛋白（F-actin）结合结构域与tarp介导的侵袭效率有关。野生型沙眼衣原体进入HeLa细胞导致宿主细胞形状改变，而tarP突变体则没有。最后，利用一种新的顺式互补方法，缺乏tarP的沙眼衣原体在小鼠生殖道感染模型中表现出显著的衰减。总之，这些数据为Tarp f -actin结合结构域在宿主细胞侵袭中的关键作用以及Tarp效应物作为真正的沙眼衣原体毒力因子提供了明确的遗传证据。
The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia's ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.