Application of β-lactamase reporter fusions as an indicator of effector protein secretion during infections with the obligate intracellular pathogen Chlamydia trachomatis.
Application of β-lactamase reporter fusions as an indicator of effector protein secretion during infections with the obligate intracellular pathogen Chlamydia trachomatis.
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DOI：
10.1371/journal.pone.0135295
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Fields KA
中科院分区：
文献类型：
--
作者：
Mueller KE;Fields KA
衣原体属物种利用多种分泌系统，包括III型分泌系统（T3 SS），将宿主相互作用效应蛋白部署到受感染的宿主细胞中。分泌蛋白的阐明传统上需要在替代T3 SS中异位表达，然后通过内源性候选效应物的免疫定位来确认衣原体的分泌。转化衣原体和实现重组基因产物稳定表达的能力使得能够更直接地评估分泌。我们采用TEM-1 β-内酰胺酶作为报告系统来评估衣原体蛋白分泌。我们提供的证据表明，该系统有助于可视化的分泌感染的情况下。具体来说，我们的研究结果提供了明确的证据，C。沙眼衣原体CT695在感染期间分泌。后续间接免疫荧光研究证实了CT695分泌，并表明这种效应可以在衣原体发育周期的多个点分泌。我们的研究结果表明，BlaM融合报告基因检测将允许有效地识别新的分泌蛋白。此外，这种方法可以很容易地适应，使衣原体分泌过程的更复杂的研究。
Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia.