Plasmid DNA Delivery by D-Alanine-Deficient Listeria monocytogenes
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- 作者：Benjamin E. Simon ; Noel Ybarra ; Aimé ; e O. Bonneval ; Ronald A. Barry
- 刊名：Biotechnology Progress
- 出版年：2006
- 出版时间：October 2006
- 年：2006
- 卷：22
- 期：5
- 页码：1394 - 1399
- 全文大小：290K
- 年卷期：v.22,no.5(October 2006)
- ISSN：1520-6033
文摘
Optimal DNA vaccine efficacy requires circumventing several obstacles, including lowimmunogenicity, 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 plasmidpurification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvantin vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as aDNA delivery vehicle in vitro. First, the D-alanine-deficient (
dal-dat) L. monocytogenes strainDP-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 theCMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with thetransformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absenceof D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infectionwith DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alaninewas omitted. Although transfection efficiency initially increased as a result of D-alaninesupplementation, high concentration or long-term supplementation led to sustained bacterialgrowth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficientDNA delivery by transformed bacteria must balance bacterial invasion and survival with targetcell health and survival.
dal-dat) L. monocytogenes strainDP-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 theCMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with thetransformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absenceof D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infectionwith DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alaninewas omitted. Although transfection efficiency initially increased as a result of D-alaninesupplementation, high concentration or long-term supplementation led to sustained bacterialgrowth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficientDNA delivery by transformed bacteria must balance bacterial invasion and survival with targetcell health and survival.