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Applied and Environmental Microbiology, January 2000, p. 383-391, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Use of Green Fluorescent Protein To Tag Lactic Acid Bacterium Strains under Development as Live Vaccine Vectors

Marie-Claude Geoffroy,1 Cyril Guyard,1 Brigitte Quatannens,2 Sonia Pavan,1 Marc Lange,1 and Annick Mercenier1,*

Département de Microbiologie des Ecosystèmes, Institut Pasteur de Lille,1 and UMR 3586, Institut de Biologie de Lille,2 Lille Cedex 59019, France

Received 14 July 1999/Accepted 26 October 1999

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.

* Corresponding author. Mailing address: Département de Microbiologie des Ecosystèmes, Institut Pasteur de Lille, 1, Rue du Pr. Calmette, B.P. 245, F59019 Lille Cedex, France. Phone: (33) 320-87-71-22. Fax: (33) 320-87-79-08. E-mail: annick.mercenier{at}pasteur-lille.fr.

Applied and Environmental Microbiology, January 2000, p. 383-391, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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