Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of : Analysis in Cell Culture and Murine Infection Models.

is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-) based vector (pPL2CBR) expressing codon optimized CBR- under the control of a highly expressed Listerial promoter (P) for and have compared this to a -based system expressing bacterial luciferase for BLI of the pathogen using growth experiments and models. The CBR- plasmid stably integrates into the chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR- labeled emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, -labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR- labeled bacteria was more efficient than the signal from -labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR- is suited to BLI approaches and demonstrated enhanced sensitivity relative to in the context of infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to and may have significant applications in the analysis of pathogenesis.