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

Abstract

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 (pPL2CBRopt) expressing codon optimized CBR-luc under the control of a highly expressed Listerial promoter (PHELP) 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.

Combination PD-1 and PD-L1 Blockade Promotes Durable Neoantigen-Specific T Cell-Mediated Immunity in Pancreatic Ductal Adenocarcinoma

“…See also Figure S4. (legend continued on next page) Smith et al, 2017;Stromnes et al, 2015;Ur Rahman et al, 2017). CB induces priming, intratumoral accumulation, and rapid intratumoral dysfunction of CB-specific T cells in PDA.…”

Methionyl-tRNA synthetase inhibitor has potentin vivoactivity in a novelGiardia lamblialuciferase murine infection model

“…Earlier studies had demonstrated that Fluc, Luc2, and CBR had higher sensitivity than PpyRE9h to report promoter activity in other systems. 21, 22 Hence, these red luciferases were assayed in Giardia .…”

A novel universal nano-luciferase-involved reporter system for long-term probing food-borne probiotics and pathogenic bacteria in mice by in situ bioluminescence imaging

“…Animals received substrates and bacteria by gastric inoculation before imaging, furimazine (1/20 dilution of Nano-Glo® Luciferase substrate, 200 mL gavage volume, Promega) is the substrate for NLuc system and D-luciferin (150 mg kg À1 , PerkinElmer) is for FLuc and CBRLuc systems. 35,36 Bioluminescence images were acquired at room temperature on a Berthold NightOWL LB 983 Imaging System (Berthold Technologies, Bad Wildbad, Germany). The images were acquired with open lter and exposure time ranges from 1 to 3 min according to the signal intensity, the luminescence counts acquired under different exposure time were all normalized as the average value (total counts/ exposure time) to ensure the reasonable comparison, and the pseudo colour images represented the light intensity (from red for most intense to blue for least intense).…”

“…S1 ‡), which was consistent with previously-reported results. 36 The short wavelength of luciferase system may limit its application in vivo, due to the weak ability to penetrate host tissues compared with long emission systems. 37 The emission maximum of NLuc was 460 nm, which was shorter than FLuc (560 nm) and CBRLuc (613 nm).…”