Novel Fluorescent Reporters for Studying Pathogen-host Interactions

Fluorescent proteins, such as green fluorescent protein (GFP), have diverse applications in the basic and applied sciences. While GFP has been frequently used in eukaryotic systems, its applications have been limited in microorganisms due to a lack of broad-range molecular tools. In this study, we have developed a vector to express GFP in pathogenic bacteria for use in bacterial pathogenesis and pathogen-host studies. A shuttle vector encoding the GFP variant mut31 (pUCP18-MCSgfpmut3) was generated and successfully transformed into various Gram-negative opportunistic pathogens from the ATCC collection, including: Escherichia coli (ATCC 25922), Salmonella enterica (ATCC 14028), Shigella flexneri (ATCC 12022), Pseudomonas aeruginosa (ATCC 10145), and the P. aeruginosa type strain PAO1 (ATCC 15692). P. aeruginosa was used as a model to test the characteristics of the vector and sensitivity of detection using a fluorescence plate reader, microscopy, flow cytometry, and in vivo imaging systems.