Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of Listeria monocytogenes

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule for monitoring in vivo gene expression in eukaryo tic and prokaryotic cells. We constructed a series of GFP vectors for in si tu detection of the intracellular pathogen Listeria monocytogenes. The gfp- mut1 gene, which encodes a red-shifted GFP, was transcriptionally fused to a strong L. monocytogenes promoter and inserted into various Escherichia co li-Listeria shuttle vectors: i) the integrative monocopy plasmid pAT113; ii ) the low copy number plasmid pTCV-Ex1; iii) the high copy number plasmid p AT18. Listeria cells harboring pNF6 and pNF7, constructed from pAT113 and p TCV-Ex1, respectively, gave low fluorescence intensities, and were opticall y detected in cultured macrophages, but not in tissue sections. The fluores cence of Listeria with the pAT18 derivative pNF8 was about 40 times greater than that with pNF6 and 15 times greater than that with pNF7. Listeria cel ls harboring pNF8 were readily detected in both cultured macrophages and ti ssue sections. Constructed GFP vectors did not affect the virulence of L. m onocytogenes in a murine model of infection. (C) 2000 Editions scientifique s et medicales Elsevier SAS.