AUTHOR=Ur Rahman Sadeeq , Stanton Michael , Casey Pat G. , Spagnuolo Angela , Bensi Giuliano , Hill Colin , Francis Kevin P. , Tangney Mark , Gahan Cormac G. M. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.01797

DOI=10.3389/fmicb.2017.01797

ISSN=1664-302X

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