The Genetic Transformation of Chlamydia pneumoniae
The Genetic Transformation of Chlamydia pneumoniae
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DOI：
10.1128/msphere.00412-18
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发表时间：
2018-09-01
期刊：
影响因子：
4.8
通讯作者：
Rupp, Jan
中科院分区：
文献类型：
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作者：
Shima, Kensuke;Wanker, Maximilian;Rupp, Jan
我们展示了使用产生稳定转化体的质粒穿梭载体系统对肺炎衣原体进行遗传转化。马肺炎衣原体 N16 分离株含有 7.5 kb 质粒 pCpnE1。我们构建了包含 pCpnE1 主链、红移绿色荧光蛋白 (RSGFP) 以及氯霉素乙酰转移酶 (CAT) 基因的质粒载体 pRSGFPCAT-Cpn，用于选择带有质粒穿梭载体的肺炎衣原体转化体。使用 pRSGFPCAT-Cpn 质粒构建体，证明了考拉分离株肺炎衣原体 LPCoLN 中 RSGFP 的表达。此外，我们发现人类心血管分离株肺炎衣原体CV-6和人类社区获得性肺炎相关肺炎衣原体IOL-207也可以用pRSGFPCAT-Cpn转化。之前的研究表明，衣原体属。当质粒穿梭载体是由与同源物种不同的质粒主链构建时，不能被转化。因此，我们证实pRSGFPCAT-Cpn不能跨越带质粒和无质粒的沙眼衣原体、鼠衣原体、豚鼠衣原体、白皮衣原体和流产衣原体中的物种屏障。然而，与我们的预期相反，pRSGFPCAT-Cpn 确实转化了猫科动物。此外，pRSGFPCAT-Cpn 不与 C. felis 的野生型质粒重组。总而言之，我们首次为肺炎衣原体提供了一种易于操作的转化方案，可产生稳定的转化体。此外，该载体可以跨越物种屏障到达猫衣原体，这表明通过质粒进行水平致病基因转移的潜力。 重要性 肺炎衣原体遗传操作工具的缺乏阻碍了对其生物学各个方面的研究。在本研究中，我们建立了一种基于质粒穿梭载体系统的肺炎衣原体转化的新型可重复方法。我们构建了肺炎衣原体质粒骨干穿梭载体pRSGFPCAT-Cpn。该构建体表达与肺炎衣原体中氯霉素乙酰转移酶融合的红移绿色荧光蛋白（RSGFP）。即使在不存在氯霉素的情况下，肺炎衣原体转化体也能稳定保留 pRSGFPCAT-Cpn 并在上皮细胞中表达 RSGFP。使用 pRSGFPCAT-Cpn 对肺炎衣原体的成功转化将推动衣原体遗传学领域的发展，并且是研究肺炎衣原体生物学中基因功能的一种有前途的新方法。此外，我们证明pRSGFPCAT-Cpn克服了质粒物种障碍，无需与内源质粒重组，表明衣原体物种之间的质粒水平衣原体致病基因转移的潜在可能性。
We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate C. pneumoniae LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate C. pneumoniae CV-6 and the human community-acquired pneumonia-associated C. pneumoniae IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that Chlamydia spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free C. trachomatis, C. muridarum, C. caviae, C. pecorum, and C. abortus. However, contrary to our expectation, pRSGFPCAT-Cpn did transform C. felis. Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of C. felis. Taken together, we provide for the first time an easy-to-handle transformation protocol for C. pneumoniae that results in stable transformants. In addition, the vector can cross the species barrier to C. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCE The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniae. C. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneu-moniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.