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Listeria monocytogenes  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- luc ) based vector (pPL2CBR opt ) expressing codon optimized CBR- luc  under the control of a  h ighly  e xpressed  L isterial  p romoter (P HELP ) for  L. monocytogenes  and have compared this to a  lux -based system expressing bacterial luciferase for BLI of the pathogen using  in vitro  growth experiments and  in vivo  models. The CBR- luc  plasmid stably integrates into the  L. monocytogenes  chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR- luc  labeled  L. monocytogenes  emits a bright signal in exponential phase that is maintained during stationary phase. In contrast,  lux -labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR- luc  labeled bacteria was more efficient than the signal from  lux -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- luc  is suited to BLI approaches and demonstrated enhanced sensitivity relative to  lux  in the context of  Listeria  infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to  lux  and may have significant applications in the analysis of  L. monocytogenes  pathogenesis.

frontiers in microbiology

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