title: Chromosomal gfp labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon: application for studies of bacteria-host interactions.
abstract: Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.
authors.affiliation: Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B5E1, Canada.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Barnes
authors.forename: Kam Tin
authors.initials: KT
authors.lastname: Leung
authors.forename: Heidi
authors.initials: H
authors.lastname: Schraft
authors.forename: Marina
authors.initials: M
authors.lastname: Ulanova
mesh_terms: D001422:Bacterial Adhesion; D002460:Cell Line; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D004847:Epithelial Cells / Q000382:microbiology; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000235:genetics* / Q000502:physiology
chemical_list: D004251:DNA Transposable Elements; D005353:Fibronectins; D049452:Green Fluorescent Proteins