Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery

Abstract

Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approach to express plasmidencoded heterologous proteins (protein antigens, toxins or enzymes) in a large set of different cell types including phagocytic and nonphagocytic mammalian cells. Previously, we have described a Listeria monocytogenes-mediated DNA delivery system, which releases plasmid DNA directly into the cytosol of mammalian cells by partial self-destruction of the carrier bacteria. Here we report on a second generation of this phage lysin supported bactofection system, which is greatly improved with respect to plasmid stability, transfer efficacy and biosafety. In this case, DNA release is initiated by spontaneous bacterial lysis in the infected cells cytosol which is subsequently enhanced by the simultaneously released phage lysin produced by the intracellular carrier bacteria. Bacteria that are capable of cell-to-cell spread are found to be much more efficient in bactofection than their nonspreading counterparts.

Cancer immunotherapy using recombinantListeria monocytogenes: Transition from bench to clinic

“…Although the expressed phage lysin remains in the cytoplasm of the producing carrier bacterium and does not lyse all intracellular bacteria, the extent of lysis is found to be sufficient to release DNA. 40 With this method of delivery the efficiency of bactofection of several phagocytic and nonphagocytic mammalian cells using L. monocytogenes is found to be comparable to other reported bactofection systems. Souders et al created a L. monocytogenes DNA delivery vaccine utilizing similar approach based on DNA delivery of a TAA with suicidal plasmid.…”

“…40 This suicidal L. monocytogenes was created using an attenuated backbone that contains deletions in the genes for the synthesis of aromatic amino acid, aroA and tryptophanyl tRNA synthetase, trpS and utilizes a plasmid that encodes for a lysis cassette to lyse the bacterial cell wall for DNA delivery. 32 The lysis of carrier L. monocytogenes takes place due to the expression of phage lysin 118 once the bacteria has entered the cytosol of the infected mammalian cells.…”

Delivery of functional DNA and messenger RNA to mammalian phagocytic cells by recombinant yeast

“…Accordingly, DNA/RNA vaccination has become the most promising strategy for inducing cellmediated immunity. [1][2][3] To do so, several approaches have been described reaching from classical DNA vaccination up to the delivery of antigenencoding DNA or RNA by viruses or virulence-attenuated bacteria. Among these bacteria, most of the strains that have been used for antigen delivery belong to the large group of invasive enteric bacterial pathogens such as Escherichia coli, Shigella, Salmonella or Listeria.…”

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli

“…Proof-of-principle for bactofection in a range of non-phagocytic cell lines, including airway epithelial cells, has been established in vitro. 13,14,[17][18][19] These studies have mainly focused on transfer of the GFP reporter gene. However, more relevant to our work, recombinant E. coli and L. monocytogenes were recently used to transfer artificial chromosomes carrying the CFTR gene locus 20 or a pCMV-CFTR plasmid, 10,21 respectively, to cell lines in vitro.…”