Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer.

Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice.

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a ß-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli.

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM ß-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of ß-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. ß-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant ß-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular ß-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly ß-CGTase by the recombinant E. coli.

Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells.

BACKGROUND: Lactobacillus plantarum, a major species of Lactic Acid Bacteria (LAB), are capable of producing postbiotic metabolites (PM) with prominent probiotic effects that have been documented extensively for rats, poultry and pigs. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on cytotoxic and antiproliferative activity of PM produced by L. plantarum. Therefore, the cytotoxicity of PM produced by six strains of L. plantarum on various cancer and normal cells are yet to be evaluated. METHODS: Postbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses. RESULTS: UL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis. CONCLUSION: In conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer.