Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors

The lactic acid bacteria (LAB) are safe microorganisms which are mainly use d for the preparation of fermented foods and for probiotic applications, Th e potential of LAB as live vehicles for the production and delivery of ther apeutic molecules such as antigens is also being actively investigated toda y. However, very little is known about the fate of live LAB when administer ed 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 micr oflora and to unravel the mechanisms underlying the postulated health-benef icial effect. We therefore started to investigate both aspects in a mouse m odel with two LAB species presently under development as live vaccine vecto rs, i.e., Lactococcus lactis and Lactobacillus plantarum. We have construct ed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this v isible marker was best expressed when placed under the control of the induc ible strong nisA promoter from L. lactis. Notably, a threshold amount of GF P was necessary to obtain a bright fluorescent phenotype. We further demons trated 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 trac e it in the gastrointestinal tract of mice upon oral administration.