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Appl Environ Microbiol. May 2007; 73(9): 3091–3094.
Published online Mar 9, 2007. doi:  10.1128/AEM.02940-06
PMCID: PMC1892880

Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro[down-pointing small open triangle]

Abstract

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.

Bioluminescent imaging (BLI) has been applied recently to a number of pathogens, including Escherichia coli (2, 23, 25, 27), Salmonella enterica serovar Typhimurium (1, 4, 6), Citrobacter rodentium (17), Staphylococcus aureus (9), and Streptococcus pneumoniae (10). Recently, in vivo murine BLI of Listeria monocytogenes 10403S was reported (13). However, the method by which this strain has been tagged has a number of drawbacks for general application. Firstly, the luciferase genes were introduced via a transposon; thus, a bank of transposon integrants must be screened for high levels of light. Secondly, the strain with the highest levels of light had the transposon integrated into the promoter region of the flaA gene. However, the flaA gene in 10403S is deregulated (12), and the same light levels are not observed in other strains (see below). Lastly, the selected clone (L. monocytogenes 2C) was highly luminescent but had a fourfold-higher 50% lethal dose than did the parental strain 10403S (13). We therefore sought to develop a method to reproducibly tag different strains of L. monocytogenes with lux by using constitutively expressed luminescence.

Recently, our group developed pPL2lux, a chromosomal integration vector containing a synthetic lux operon derived from Photorhabdus luminescens and optimized for use with gram-positive bacteria (20), to monitor gene expression in L. monocytogenes (3). Here, this promoter probe vector was used to test a range of promoters for high and constitutive expression in L. monocytogenes.

PCP25 is a highly active, constitutive lactococcal consensus promoter whose sequence has been published (14). PCP25 was synthesized by “gene tiling,” as described previously (15). Basically, PCR was performed with KOD hotstart DNA polymerase (Merck, Nottingham, United Kingdom), using 40-mer oligonucleotides with consecutive overlaps of 20 bases and covering the entire sequence of the promoter as template. Phelp (for “highly expressed Listeria promoter” [Fig. [Fig.1A])1A]) was generated by introducing the 5′ untranslated region of the L. monocytogenes EGDe hlyA gene (11) into PCP25 by the same gene tiling approach. Promoters for the listerial genes ldh, p60, and flaA were amplified from EGDe genomic DNA by PCR (~500 bp upstream and including the start codon [http://genolist.pasteur.fr/ListiList/]). All promoters were cloned into pPL2lux as exact translational fusions in front of luxABCDE. The resulting constructs (pPL2luxPCP25, pPL2luxPhelp, pPL2luxPldh, and pPL2luxPp60), as well as the previously described pPL2luxPsecA and pPL2luxPhlyA (3), were integrated into a tRNAArg gene in the chromosome of EGDe (11) by the site-specific integration system of pPL2 (16). Successful integration was indicated by a chloramphenicol resistance phenotype and colony PCR, as described previously (3). Growth of the different strains was compared by monitoring the optical density at 600 nm (OD600) in 96-well plates with a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). Luminescence was measured in relative light units (RLU) (in photons s−1) in a Xenogen IVIS100 (Xenogen, Alameda, CA). No significant difference in growth rate or final OD600 was observed for any of the strains harboring a pPL2lux construct compared to the wild-type strain (Fig. (Fig.1B).1B). Plasmid integration was stable for all constructs in the absence of antibiotic for at least 50 generations. All listerial promoters gave luminescence during growth in brain heart infusion broth (BHI) broth (Fig. (Fig.1C)1C) and LB (data not shown). Of the native listerial promoters tested, Pldh gave highest luminescence. Pldh was selected based on preliminary microarray data in which ldh was the most highly expressed gene in EGDe grown in BHI broth (unpublished data). PCP25 gave rise to luminescence comparable to that observed with the most highly expressing listerial promoters. Phelp showed a 62- to 170-fold increase in luminescence compared to PCP25 throughout growth in BHI broth (Fig. (Fig.1C).1C). This is in agreement with a recent study in which the introduction of the hlyA 5′ untranslated region into HyperSPO1, a constitutive Bacillus subtilis promoter (21), resulted in approximately 50-fold-higher levels of gfp expression (22).

FIG. 1.
(A) Sequence and important features of Phelp. The sequence portion derived from PCP25 is shaded in gray. The XhoI restriction site, the −35 and −10 regions, the ribosome binding site (RBS), and the translational start are boxed; transcription ...

Constitutive expression of luminescence with Phelp was further tested in hot dogs and Camembert, two food products associated with listeriosis outbreaks (7, 24). For this purpose, hot dog or Camembert samples (20 g) were thoroughly homogenized in brine or phosphate-buffered saline (PBS) (10 ml) with a tissue grinder and then inoculated with wild-type EGDe or EGDe::pPL2luxPhelp (hereafter termed EGDelux) in PBS (10 ml) to give a final bacterial load of 1 × 107 CFU per g. These homogenates were then incubated at 37°C, and bacterial growth was monitored by CFU per gram and RLU. EGDe grew well in hot dog homogenate (Fig. (Fig.2),2), and during exponential growth (t = 0 to 5 h), CFU closely correlated with RLU (R2 = 0.989; P = 4.6 × 10−5). In Camembert homogenate, no significant growth of EGDe could be observed by CFU (although high standard errors were recorded). By contrast, with EGDelux, good luminescence in Camembert homogenates was observed throughout the experiment (Fig. (Fig.2A).2A). Quantification of luminescence was more reproducible than CFU and indicated a twofold increase in bacterial load in Camembert homogenate (Fig. (Fig.2B2B [t of 1 h compared to t of 7 h]). Similar results were observed when food homogenates were stored at 4°C and growth and luminescence were monitored over a 6-day period (data not shown). These bioluminescently tagged Listeria cells may offer a rapid and simple method for establishing whether food products are at risk of supporting Listeria growth, thus supporting the new U.S. guidelines (8).

FIG. 2.
Luminescence imaging of hot dog and Camembert. (A) Homogenates inoculated with EGDe or EGDelux. The color bar indicates bioluminescence signal intensity (in photons s−1 cm−2). (B) Comparison of RLU (bars) and CFU (symbols) from hot dog ...

The use of EGDelux for in vivo imaging was also tested in conventional female BALB/c mice. Animals were kept in a conventional animal colony, and all experiments were approved by the animal ethics committee of University College Cork. Mice were inoculated with 1 × 104 CFU of washed overnight cultures in 100 μl of PBS by injection into the tail vein. Three days postinoculation, the animals were anesthetized with isoflurane and imaged in a Xenogen IVIS 100 system. Systemic infection of mice with EGDelux was observed at day 3 of infection. Two distinct signals of luminescence were observed (Fig. (Fig.3A)3A) and, after dissection, could be assigned to livers and spleens (Fig. (Fig.3A),3A), the major sites of listerial infections in mice. Bacterial numbers recovered from the organs of mice infected with EGDelux (8.0 ± 0.2 log10 CFU/spleen and 8.0 ± 0.4 log10 CFU/liver; n = 4 [per organ]) were not significantly different from those of mice infected with wild-type EGDe (7.6 ± 0.7 log10 CFU/spleen and 7.5 ± 1.2 log10 CFU/liver; n = 4 [per organ]). Moreover, quantification of luminescence signals from dissected organs of mice infected with EGDelux revealed a close correlation with CFU (livers: R2 = 0.994, P = 2.8 × 10−11; spleens: R2 = 0.985, P = 1.4 × 10−9; n = 11).

FIG. 3.
(A) In vivo BLI of a mouse infected with EGDelux (day 3 postinfection) and postmortem-dissected liver and spleen. (B) In vivo BLI of a mouse injected intratumorally with EGDelux (day 3 postinjection) and postmortem-dissected liver, spleen, and tumor. ...

It is well documented that many bacterial species can enter and replicate in solid tumors in animals, representing a powerful approach for specifically locating metastatic tumors and acting as vectors for therapeutic gene delivery (26, 28). Due to its intracellular lifestyle, L. monocytogenes is an attractive candidate for bactofection delivery of plasmids carrying therapeutic eukaryotic genes to tumors (19). For examining the progression of infections in live animals with implanted tumors, a mechanism of imaging the trafficking and persistence of L. monocytogenes gene vectors is extremely valuable. We therefore sought to investigate whether Phelp-driven luminescence can be used to trace L. monocytogenes in tumors. Murine 4T1 mammary tumor cells (ATCC CRL-2539) were maintained according to ATCC recommendations. Subcutaneous tumors were induced in the flanks of female BALB/c mice as described previously (5), with 7 × 103 4T1 cells. Animals with tumors of ~100 mm3 in size received intratumoral injections of 1 × 104 CFU in 100 μl of PBS. EGDelux was detected throughout the duration of the experiment, i.e., until day 3, when the animals were sacrificed (Fig. (Fig.3B).3B). By dissecting the tumors it could be confirmed that the luminescent signal from the injection site came from the tumor itself (Fig. (Fig.3B).3B). The RLU detected from the tumors in living animals at days 0, 1, and 2 did not differ significantly from the RLU detected at day 3 (data not shown). This indicates that EGDelux remained viable within the tumors without a significant decrease in bacterial load. Additionally, at day 3 a signal from the area of the spleen was detected, indicating that the bacteria had spread from the tumors, causing systemic infection. Systemic infection was confirmed by bioluminescent signals from dissected livers and spleens (Fig. (Fig.3B3B).

In L. monocytogenes 2C (13), the lux-tagged version of strain 10403S, the transposon was found to have integrated into the promoter region of the flaA gene, whose expression was shown to be deregulated in strain 10403S (12). Translational fusion of the flaA promoter to luxABCDE in pPL2lux and subsequent integration into the chromosome revealed that the maximum levels of luminescence in BHI broth at 37°C were (9.32 ± 0.14) × 105 RLU for 10403S::pPL2luxPfla, while no luminescence above background was observed for EGDe::pPL2luxPflaA ([<3.05 ± 0.43] × 103 RLU). This is consistent with previous observations of flaA expression in different strains of L. monocytogenes (12). To test whether Phelp was functional in other strains of Listeria, pPL2luxPhelp was successfully integrated into the chromosomes of strains 10403S and F2365 (18). The resulting strains, 10403Slux and F2365lux, showed levels of luminescence almost identical to that of EGDelux throughout growth in BHI broth (maximal luminescence at the end of exponential growth, [2.96 ± 0.09] × 106 RLU for EGDelux, [2.69 ± 0.04] × 106 RLU for 10403Slux, and [2.72 ± 0.16] × 106 RLU for F2365lux).

In conclusion, we have developed a system for the stable constitutive expression of high levels of luminescence in L. monocytogenes. In combination with the single-copy site-specific integration of pPL2, Phelp-driven expression of luxABCDE was used to monitor L. monocytogenes in food matrices, in systemic infections in vivo, and for tumor targeting. pPL2luxPhelp offers the possibility of labeling different strains and mutants of L. monocytogenes with the same efficient expression of luminescence without the need for extensive screening of transposon mutant libraries.

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 March 2007.

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