Development of a Fluorescent Tool for Studying Intracellular Infection.

Legionnaires' disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Nominally a parasite of protozoa, can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. pathogenesis has been well characterized; however, little is known about the >25 different spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an clinical isolate using approaches previously established for investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the presence in the protozoan infection model. As comparative controls, strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating non-pneumophila spp. in real time.