title: In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A.
abstract: BACKGROUND
  The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant invasive LL expressing Fibronectin Binding Protein A of Staphylococcus aureus (LL-FnBPA+) or Internalin A of Listeria monocytogenes (LL-InlA+), were able to deliver and trigger DNA expression by epithelial cells, either in vitro or in vivo. InlA does not bind to its receptor, the murine E-cadherin, thus limiting the use of LL-InlA+ in in vivo murine models. Moreover, FnBPA binds to its receptors, integrins, via fibronectin introducing another limiting factor. In order to avoid the limitations of LL-InlA+ and LL-FnBPA+, a new L. lactis strain was engineered to produce a previously described mutated form of InlA (LL-mInlA+) allowing the binding of mInlA on murine E-cadherin.
  
  
  RESULTS
  After showing the expression of mInLA at the surface of LL-mInlA+ strain, in vitro gentamycin survival assay in Caco-2 cells showed that LL-mInlA+ is 1000 times more invasive than LL. LL-mInlA+ invasivity was also validated by fluorescence microscopy. LL and LL-mInlA+ were transformed with pValacBLG, a plasmid containing the cDNA of bovine β-Lactoglobulin (BLG), resulting in strains LL-BLG and LL-mInlA+BLG. The plasmid transfer in vitro using LL-mInlA+BLG was increased 10 times compared to LL-BLG. Moreover, the number of mice producing BLG in isolated enterocytes after oral administration of LL-mInlA+BLG in vivo was slightly higher than after oral administration of LL-BLG.
  
  
  CONCLUSIONS
  We confirmed in this study that the production of mInlA at the surface of L. lactis is a promising strategy for plasmid transfer in vitro and in vivo.
authors.affiliation: INRA, UMR1319 Micalis, Commensals and Probiotics-Host Interactions Laboratory, Jouy-en-Josas, France.
authors.forename: Marcela
authors.initials: M
authors.lastname: de Azevedo
authors.forename: Jurgen
authors.initials: J
authors.lastname: Karczewski
authors.forename: François
authors.initials: F
authors.lastname: Lefévre
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
authors.forename: Anderson
authors.initials: A
authors.lastname: Miyoshi
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Wells
authors.forename: Philippe
authors.initials: P
authors.lastname: Langella
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chatel
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018938:Caco-2 Cells; D004705:Endocytosis; D020895:Enterocytes / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D007782:Lactoglobulins / Q000378:metabolism; D008089:Listeria monocytogenes / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007782:Lactoglobulins; D011994:Recombinant Proteins; D019444:Vaccines, DNA; C069761:internalin protein, Bacteria