title: Plasmid DNA delivery by D-alanine-deficient Listeria monocytogenes.
abstract: Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the D-alanine-deficient (Deltadal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of D-alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alanine was omitted. Although transfection efficiency initially increased as a result of D-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.
authors.affiliation: Department of Veterans Affairs Medical Center, Portland, Oregan, USA. simonb@evergreen.edu
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Simon
authors.forename: Noel
authors.initials: N
authors.lastname: Ybarra
authors.forename: Aimée O
authors.initials: AO
authors.lastname: Bonneval
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Barry
mesh_terms: D000409:Alanine / Q000737:chemistry*; D000818:Animals; D001709:Biotechnology / Q000379:methods; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008089:Listeria monocytogenes / Q000378:metabolism*; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA; D000409:Alanine