Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.
Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.
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在研究宿主 - 微生物相互作用中，底部厌氧菌的遗传转化，用于FMN绿色荧光蛋白表达。
DOI：
10.1371/journal.pone.0018499
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发表时间：
2011-04-15
期刊：
影响因子：
3.7
通讯作者：
Yilmaz Ö
中科院分区：
文献类型：
--
作者：
Choi CH;DeGuzman JV;Lamont RJ;Yilmaz Ö
最近引入的“氧非依赖性”黄素单核苷酸（FMN）为基础的荧光蛋白（FbFPs）的主要兴趣，真核和原核微生物生物学家。因此，我们第一次证明了专性厌氧菌，口腔的成功机会致病菌牙龈卟啉单胞菌，可以被基因工程改造用于表达无毒的绿色FbFP。所得的转化体对于研究活宿主细胞中的动态细菌过程是有功能的。转化的牙龈卟啉单胞菌（PgFbFP）的可视化显示了强荧光，如通过荧光显微镜和荧光分光光度法测定的，其在495 nm处达到最大发射。用PgFbFP感染人原代牙龈上皮细胞（GECs），通过荧光定量显微镜和抗生素保护试验分析细菌对宿主细胞的侵袭。结果显示野生型和PgFbFP菌株的细胞内细菌水平相似。结合细胞器特异性荧光染料，利用转化的菌株提供了对活的/代谢活性的牙龈卟啉单胞菌随时间在GEC中的运输的直接和准确的测定。此外，GECs共感染PgFbFP和ATP依赖性Clp丝氨酸蛋白酶缺陷型突变体（ClpP-），以研究两种菌株在同一宿主细胞内的差异命运。定量共定位分析显示细胞内PgFbFP与内质网网络显著相关，而大多数ClpP-生物体被运输到溶酶体中。因此，我们已经开发了一种新的和可靠的方法来表征活的宿主细胞-微生物相互作用，并证明了FMN-绿色荧光蛋白的适应性研究专性厌氧生物体诱导的持续性宿主感染。
The recent introduction of “oxygen-independent” flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP) revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs) were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP-) to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms.
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