A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays

Abstract

Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.

Recombinant Invasive Lactococcus lactis Carrying a DNA Vaccine Coding the Ag85A Antigen Increases INF-γ, IL-6, and TNF-α Cytokines after Intranasal Immunization

“…For this last application it was shown that both, native and invasive L. la ctis, can be used as in vitro and in vivo DNA delivery vehicles for plasmid transfer into mammalians cells (Guimaraes et al, 2006; Chatel et al, 2008; Innocentin et al, 2009; Almeida et al, 2014; Mancha-Agresti et al, 2016). …”

Bacillus subtilis expressing dsVP28 improved shrimp survival from WSSV challenge

“…The B. subtilis bactofection mechanism in shrimp could be similar to the Lactococcus lactis, a gram-positive lactic acid bacterium, bactofection mechanism in mice [38]. B. subtilis probably adheres to epithelial cells and enters the cell by internalization of a vacuole/phagosome.…”

Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases Related to the GIT

“…New vectors had been developed to approach the DNA vaccine using LAB as live delivery vehicles [146,147,[148][149][150]. These vectors present a series of common characteristics such as the presence of a eukaryotic promoter, which allows protein expression by eukaryotic cells; a prokaryotic region, which has a selection marker (usually antibiotic resistance); a multiple cloning site, where the open reading frame (ORF) of interest will be inserted; and a prokaryotic origin of replication, which ensures that the plasmid replicates only in prokaryotic cells [151].…”

“…These vectors present a series of common characteristics such as the presence of a eukaryotic promoter, which allows protein expression by eukaryotic cells; a prokaryotic region, which has a selection marker (usually antibiotic resistance); a multiple cloning site, where the open reading frame (ORF) of interest will be inserted; and a prokaryotic origin of replication, which ensures that the plasmid replicates only in prokaryotic cells [151]. Some molecules (IL-10, IL-4, and HSP65) have been cloned in these vectors to evaluate their effect, especially as a treatment approach in diseases related to the bowel [152,153], as well as reporters (GFP and Cherry) which allowed the understanding of this platform in the mammalian body [148,154]. Although further studies need to be conducted in order to elucidate whether the cloning of ORFs of interest in these vectors is really effective pointing to disease prevention and treatment, this approach is undoubtedly an important tool for the development of new techniques with potential in the medical clinic.…”