Genetic Transformation of an Obligate Anaerobe, P. gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe InteractionReportar como inadecuado




Genetic Transformation of an Obligate Anaerobe, P. gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe Interaction - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

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.



Autor: Chul Hee Choi, Jefferson V. DeGuzman, Richard J. Lamont, Özlem Yilmaz

Fuente: http://plos.srce.hr/



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