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. 2006 Apr;72(4):2876-84.
doi: 10.1128/AEM.72.4.2876-2884.2006.

Novel luciferase reporter system for in vitro and organ-specific monitoring of differential gene expression in Listeria monocytogenes

Peter A Bron  1 , Ian R Monk, Sinéad C Corr, Colin Hill, Cormac G M Gahan
Affiliations

Novel luciferase reporter system for in vitro and organ-specific monitoring of differential gene expression in Listeria monocytogenes

Peter A Bron et al. Appl Environ Microbiol. 2006 Apr.

Abstract

In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes.

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Figures

FIG. 1.
FIG. 1.
Plasmid map of pPL2lux. The luxABCDE operon was derived from pSB2025 (34) with a blunt-end SwaI restriction site introduced overlapping the ATG start codon of luxA. Cloning of PCR-amplified promoter elements into the SalI and SwaI restriction sites allowed construction of exact translational fusions of listerial promoters to luxABCDE.
FIG. 2.
FIG. 2.
Hemolysin expression profiles during growth at 37°C in LB medium buffered at pH 7.4. The triangles and diamonds indicate the average growth of quadruplicate cultures and the gray and black bars indicate the average luciferase expression profiles for EGD-e::pPL2lux-PsecA and EGD-e::pPL2lux-PhlyA, respectively. BLC, bioluminescence counts; OD600nm, optical density at 600 nm. The data are representative of the data from three independent experiments. At times 1, 2, and 3, samples were taken from the cultures for RNA isolation. Subsequently, luciferase transcript levels were assessed by RT-PCR analysis (inset).
FIG. 3.
FIG. 3.
Correlation between bioluminescence and mRNA levels. EGDe::pPL2lux-PhlyA was grown in LB medium, LB medium containing 0.2% activated charcoal (LBAC), or LB medium containing 25 mM cellobiose (LBcell). Upon entry into the stationary phase, 1-ml portions of the cultures were imaged using the IVIS 100 system (A), and cDNA was synthesized from RNA extracted from 10-ml portions of the cultures, which was followed by PCR analysis to assess levels of 16S RNA, luxA, and hlyA transcription (B). The images in panel A were obtained using an IVIS 100 system with 5 min of exposure and a binning value of 8. The color bar indicates the bioluminescence signal intensity (in photons s−1 cm−2). Fold induction indicates the levels of bioluminescence relative to the levels observed in LB medium.
FIG. 4.
FIG. 4.
Hemolysin expression during cell invasion. EGDe::pPL2lux-PhlyA was used to infect monolayers of C2Bbe1 cells, and bioluminescence was monitored 0, 30, 60, and 120 min postinfection. The images were obtained using an IVIS 100 system with 5 min of exposure and a binning value of 8. BLC, bioluminescence counts (in photons s−1). The color bar indicates bioluminescence signal intensity (in photons s−1 cm−2). ND, not detectable (bioluminescence counts, <1 × 104 photons s−1). The values for bioluminescence counts and CFU are the means ± standard deviations from triplicate wells. The data are representative of the data from three independent experiments.
FIG. 5.
FIG. 5.
In vivo analysis of L. monocytogenes hemolysin expression and organ-specific colonization in BALB/c mice. Two mice were inoculated with 2 × 106 bacteria via the intraperitoneal route using either EGD-e::pPL2lux (Control) or EGD-e::pPL2lux-PhlyA (hlyA). Three days postinfection, the mice were euthanized, and bioluminescent imaging was performed for 5 min at a binning value of 4 using an IVIS 100 system. BLC, bioluminescence counts (in photons s−1). The color bar indicates the bioluminescence signal intensity (in photons s−1 cm−2). Subsequently, the numbers of bacterial CFU were determined for the individual organs. Results for the left and right kidneys were combined to obtain total BLC and CFU values. ND, not detectable (bioluminescence counts, <1 × 104 photons s−1). The data are representative of the data from three independent experiments.
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