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.
Highlights
Listeria monocytogenes is an opportunistic facultative intracellular pathogen which is capable of withstanding harsh environmental conditions, including stresses encountered during the processing, packaging and storage of foods (Gahan and Hill, 2005)
L. monocytogenes infection in mice has been used as an important model for the analysis of intracellular parasitism and subsequent immunity and this research has benefited from the development of molecular tools to analyze bacterial and host responses in vivo (Cossart and Toledo-Arana, 2008; Lebreton et al, 2016)
A codon optimized click beetle red luciferase (CBR-luc) (CBRopt) gene for optimal expression in L. monocytogenes was synthesized under the influence of a constitutive synthetic promoter, the highly expressed Listeria promoter PHELP (Riedel et al, 2007)
Summary
Listeria monocytogenes is an opportunistic facultative intracellular pathogen which is capable of withstanding harsh environmental conditions, including stresses encountered during the processing, packaging and storage of foods (Gahan and Hill, 2005). L. monocytogenes infection in mice has been used as an important model for the analysis of intracellular parasitism and subsequent immunity and this research has benefited from the development of molecular tools to analyze bacterial and host responses in vivo (Cossart and Toledo-Arana, 2008; Lebreton et al, 2016). Bioluminescence imaging (BLI) is a non-invasive technology that permits the molecular analysis of cells through the expression of proteins that emit visible light (Byrne et al, 2013). Eukaryotic luciferases from natural sources (e.g., firefly, click beetle, sea pansy) can be expressed in bacteria and may offer alternatives to the use of bacterial Lux for bacterial imaging studies (Gahan, 2012; Tangney and Francis, 2012; Chang et al, 2014; Karimi et al, 2016)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
