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.

Ultrastructural and Morphological Effects in T-Lymphoblastic Leukemia CEM-SS Cells Following Treatment with Nordamnacanthal and Damnacanthal from Roots of Morinda elliptica.

BACKGROUND: Morinda elliptica (family Rubiaceae), locally known as 'mengkudu kecil', has been used by the Malays for medicinal purposes. Anthraquinones isolated from the roots of Morinda elliptica, namely nordamnacanthal and damnacanthal, have been widely reported to exhibit anticancer and antioxidant properties in various cancer models in vitro and in vivo. AIM: This study analyzed the morphological and ultrastructural effects of damnacanthal and nordamnacanthal on T-lymphoblastic leukemia CEM-SS cells. METHOD: Light microscopy, Giemsa staining, Wright's staining, scanning electron microscopy, and transmission electron microscopy were carried out to determine apoptosis, necrosis, and ultrastructural changes that occurred within the cells. RESULTS: The outcomes showed that these compounds induced cell death by apoptosis and necrosis, specifically at higher doses of 10 and 30 µg/mL. Condensation and fragmentation of the nuclear chromatin, which further separated into small, membrane-bound vesicles known as apoptotic bodies, were observed in the nuclei and cytoplasm. The plasma membranes and cytoskeletons also showed marked morphological changes upon treatment with damnacanthal and nordamnacanthal, indicating apoptosis. CONCLUSION: Therefore, we report that damnacanthal and nordamnacanthal exhibit anticancer properties by inducing apoptosis and necrosis in CEM-SS cells, and they have potential as a drug for the treatment of T-lymphoblastic leukemia.

Lactococcus lactis secreting phage lysins as a potential antimicrobial against multi-drug resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an opportunistic Gram-positive bacterium that can form biofilm and become resistant to many types of antibiotics. The treatment of multi-drug resistant Staphylococcus aureus (MDRSA) infection is difficult since it possesses multiple antibiotic-resistant mechanisms. Endolysin and virion-associated peptidoglycan hydrolases (VAPGH) enzymes from bacteriophage have been identified as potential alternative antimicrobial agents. This study aimed to assess the ability of Lactococcus lactis NZ9000 secreting endolysin and VAPGH from S. aureus bacteriophage 88 to inhibit the growth of S. aureus PS 88, a MDRSA. METHOD: Endolysin and VAPGH genes were cloned and expressed in L. lactis NZ9000 after fusion with the SPK1 signal peptide for secretion. The recombinant proteins were expressed and purified, then analyzed for antimicrobial activity using plate assay and turbidity reduction assay. In addition, the spent media of the recombinant lactococcal culture was analyzed for its ability to inhibit the growth of the S. aureus PS 88. RESULTS: Extracellular recombinant endolysin (Endo88) and VAPGH (VAH88) was successfully expressed and secreted from L. lactis which was able to inhibit S. aureus PS 88, as shown by halozone formation on plate assays as well as inhibition of growth in the turbidity reduction assay. Moreover, it was observed that the spent media from L. lactis NZ9000 expressing Endo88 and VAH88 reduced the viability of PS 88 by up to 3.5-log reduction with Endo88 being more efficacious than VAH88. In addition, Endo88 was able to lyse all MRSA strains tested and Staphylococcus epidermidis but not the other bacteria while VAH88 could only lyse S. aureus PS 88. CONCLUSION: Recombinant L. lactisNZ9000 expressing phage 88 endolysin may be potentially developed into a new antimicrobial agent for the treatment of MDRSA infection.

Proteomic analysis revealed the biofilm-degradation abilities of the bacteriophage UPMK_1 and UPMK_2 against Methicillin-resistant Staphylococcus aureus.

OBJECTIVE: The degradation activity of two bacteriophages UPMK_1 and UPMK_2 against methicillin-resistant Staphylococcus aureus phages were examined using gel zymography. METHODS: The analysis was done using BLASTP to detect peptides catalytic domains. Many peptides that are related to several phage proteins were revealed. RESULTS: UPMK_1 and UPMK_2 custom sequence database were used for peptide identification. The biofilm-degrading proteins in the bacteriophage UPMK_2 revealed the same lytic activity towards polysaccharide intercellular adhesin-dependent and independent of Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers in comparison to UPMK_1, which had lytic activity restricted solely to its host. CONCLUSION: Both bacteriophage enzymes were involved in MRSA biofilm degradation during phage infection and they have promising enzybiotics properties against MRSA biofilm formation.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus.

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Anticancer Potential of Damnacanthal and Nordamnacanthal from Morinda elliptica Roots on T-lymphoblastic Leukemia Cells.

BACKGROUND: This study reports on the cytotoxic properties of nordamnacanthal and damnacanthal, isolated from roots of Morinda elliptica on T-lymphoblastic leukaemia (CEM-SS) cell lines. METHODS: MTT assay, DNA fragmentation, ELISA and cell cycle analysis were carried out. RESULTS: Nordamnacanthal and damnacanthal at IC50 values of 1.7 µg/mL and10 µg/mL, respectively. At the molecular level, these compounds caused internucleosomal DNA cleavage producing multiple 180-200 bp fragments that are visible as a "ladder" on the agarose gel. This was due to the activation of the Mg2+/Ca2+-dependent endonuclease. The induction of apoptosis by nordamnacanthal was different from the one induced by damnacanthal, in a way that it occurs independently of ongoing transcription process. Nevertheless, in both cases, the process of dephosphorylation of protein phosphates 1 and 2A, the ongoing protein synthesis and the elevations of the cytosolic Ca2+ concentration were not needed for apoptosis to take place. Nordamnacanthal was found to have a cytotoxic effect by inducing apoptosis, while damnacanthal caused arrest at the G0/G1 phase of the cell cycle. CONCLUSION: Damnacanthal and nordamnacanthal have anticancer properties, and could act as potential treatment for T-lymphoblastic leukemia.

Functional characterization of a new terpene synthase from Plectranthus amboinicus.

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.

Enhancement of Versatile Extracellular Cellulolytic and Hemicellulolytic Enzyme Productions by Lactobacillus plantarum RI 11 Isolated from Malaysian Food Using Renewable Natural Polymers.

Lactobacillus plantarum RI 11 was reported recently to be a potential lignocellulosic biomass degrader since it has the capability of producing versatile extracellular cellulolytic and hemicellulolytic enzymes. Thus, this study was conducted to evaluate further the effects of various renewable natural polymers on the growth and production of extracellular cellulolytic and hemicellulolytic enzymes by this novel isolate. Basal medium supplemented with molasses and yeast extract produced the highest cell biomass (log 10.51 CFU/mL) and extracellular endoglucanase (11.70 µg/min/mg), exoglucanase (9.99 µg/min/mg), ß-glucosidase (10.43 nmol/min/mg), and mannanase (8.03 µg/min/mg), respectively. Subsequently, a statistical optimization approach was employed for the enhancement of cell biomass, and cellulolytic and hemicellulolytic enzyme productions. Basal medium that supplemented with glucose, molasses and soybean pulp (F5 medium) or with rice straw, yeast extract and soybean pulp (F6 medium) produced the highest cell population of log 11.76 CFU/mL, respectively. However, formulated F12 medium supplemented with glucose, molasses and palm kernel cake enhanced extracellular endoglucanase (4 folds), exoglucanase (2.6 folds) and mannanase (2.6 folds) specific activities significantly, indicating that the F12 medium could induce the highest production of extracellular cellulolytic and hemicellulolytic enzymes concomitantly. In conclusion, L. plantarum RI 11 is a promising and versatile bio-transformation agent for lignocellulolytic biomass.

Effects of lysine and methionine in a low crude protein diet on the growth performance and gene expression of immunity genes in broilers.

Globally, the poultry industry is 1 of the most advanced livestock industries. Feed contributes to the biggest proportion (65-70%) of the production cost. Most feed ingredients in Malaysia are imported, which contributes to the high food bill annually, and alternative feed formulation may help decrease the cost of poultry feed. Feed formulation are improved to efficiently meet the dietary requirements of the broilers and 1 of the ways is by reducing the level of crude protein in the diet while supplementing essential amino acids. In this study, the effects of methionine and lysine, which are the 2 most limiting amino acids in the chicken diet, were supplemented in a low crude protein diet, and its effects on the growth and expression of immunity genes such as MUC2, SLC, GAL6, and LEAP-2 were studied. A total of 300 Cobb500 broilers were tested with 10 different dietary treatments. Experimental treatment diets consist of high, standard, and low levels of methionine and lysine in the diet with reduced crude protein. The control group consists of diet with standard levels of lysine, methionine, and crude protein as recommended for Cobb500 broilers. Ribonucleic acid was extracted from the jejunum, spleen, and liver for gene expression analysis which was performed with real-time polymerase chain reaction using SYBR Green chemistry. Results of the growth performance at 6 wk showed improved feed conversion ratio when lysine was increased by 0.2% in a low crude protein diet at 1.96 ± 0.11. Gene expression of MUC2 gene in the jejunum showed a significant increase across all experimental diets with the treatment with higher lysine in low crude protein diet with the highest increase of 3.8 times as compared with the control diet. The other genes expressed in the spleen and liver were mostly downregulated. It was concluded that supplementation of high lysine with standard methionine in a low crude protein diet performed better in terms of lowest feed conversion ratio and high upregulation of MUC2 gene.

Secretion of recombinant xylanase in Lactococcus lactis using signal peptides Usp45 and Spk1.

OBJECTIVE: The effect of two signal peptides, namely Usp45 and Spk1 on the secretion of xylanase in Lactococcus lactis was analysed. RESULTS: Xylanase was successfully expressed in Lactococcus lactis. Recombinant xylanase fused to either signal peptide Usp45 or Spk1 showed halo zone on Remazol Brilliant Blue-Xylan plates. This indicated that the xylanase was successfully secreted from the cell. The culture supernatants of strains secreting the xylanase with help of the Spk1 and Usp45 signal peptides contained 49.7 U/ml and 34.4 U/ml of xylanase activity, respectively. CONCLUSION: Although Usp45 is the most commonly used signal peptide when secreting heterologous proteins in Lactococcus lactis, this study shows that Spk1 isolated from Pediococcus pentosaceus was superior to Usp45 in regard to xylanase protein secretion.

Rapid Evaluation and Optimization of Medium Components Governing Tryptophan Production by Pediococcus acidilactici TP-6 Isolated from Malaysian Food via Statistical Approaches.

Tryptophan is one of the most extensively used amino acids in livestock industry owing to its effectiveness in enhancing the growth performance of animals. Conventionally, the production of tryptophan relies heavily on genetically modified Escherichia coli but its pathogenicity is a great concern. Our recent study demonstrated that a lactic acid bacterium (LAB), Pediococcus acidilactici TP-6 that isolated from Malaysian food was a promising tryptophan producer. However, the tryptophan production must enhance further for viable industrial application. Hence, the current study evaluated the effects of medium components and optimized the medium composition for tryptophan production by P. acidilactici TP-6 statistically using Plackett-Burman Design, and Central Composite Design. The optimized medium containing molasses (14.06 g/L), meat extract (23.68 g/L), urea (5.56 g/L) and FeSO4 (0.024 g/L) significantly enhanced the tryptophan production by 150% as compared to the control de Man, Rogosa and Sharpe medium. The findings obtained in this study revealed that rapid evaluation and effective optimization of medium composition governing tryptophan production by P. acidilactici TP-6 were feasible via statistical approaches. Additionally, the current findings reveal the potential of utilizing LAB as a safer alternative tryptophan producer and provides insight for future exploitation of various amino acid productions by LAB.

Biological control of Erwinia mallotivora, the causal agent of papaya dieback disease by indigenous seed-borne endophytic lactic acid bacteria consortium.

Dieback disease caused by Erwinia mallotivora is a major threat to papaya plantation in Malaysia. The current study was conducted to evaluate the potential of endophytic lactic acid bacteria (LAB) isolated from papaya seeds for disease suppression of papaya dieback. Two hundred and thirty isolates were screened against E. mallotivora BT-MARDI, and the inhibitory activity of the isolates against the pathogen was ranging from 11.7-23.7 mm inhibition zones. The synergistic experiments revealed that combination of W. cibaria PPKSD19 and Lactococcus lactis subsp. lactis PPSSD39 increased antibacterial activity against the pathogen. The antibacterial activity was partially due to the production of bacteriocin-like inhibitory substances (BLIS). The nursery experiment confirmed that the application of bacterial consortium W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 significantly reduced disease severity to 19% and increased biocontrol efficacy to 69% of infected papaya plants after 18 days of treatment. This study showed that W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 are potential candidate as biocontrol agents against papaya dieback disease.

Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis.

BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system. RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

Comparative Study of Extracellular Proteolytic, Cellulolytic, and Hemicellulolytic Enzyme Activities and Biotransformation of Palm Kernel Cake Biomass by Lactic Acid Bacteria Isolated from Malaysian Foods.

Biotransformation via solid state fermentation (SSF) mediated by microorganisms is a promising approach to produce useful products from agricultural biomass. Lactic acid bacteria (LAB) that are commonly found in fermented foods have been shown to exhibit extracellular proteolytic, ß-glucosidase, ß-mannosidase, and ß-mannanase activities. Therefore, extracellular proteolytic, cellulolytic, and hemicellulolytic enzyme activities of seven Lactobacillus plantarum strains (a prominent species of LAB) isolated from Malaysian foods were compared in this study. The biotransformation of palm kernel cake (PKC) biomass mediated by selected L. plantarum strains was subsequently conducted. The results obtained in this study exhibited the studied L. plantarum strains produced versatile multi extracellular hydrolytic enzyme activities that were active from acidic to alkaline pH conditions. The highest total score of extracellular hydrolytic enzyme activities were recorded by L. plantarum RI11, L. plantarum RG11, and L. plantarum RG14. Therefore, they were selected for the subsequent biotransformation of PKC biomass via SSF. The hydrolytic enzyme activities of treated PKC extract were compared for each sampling interval. The scanning electron microscopy analyses revealed the formation of extracellular matrices around L. plantarum strains attached to the surface of PKC biomass during SSF, inferring that the investigated L. plantarum strains have the capability to grow on PKC biomass and perform synergistic secretions of various extracellular proteolytic, cellulolytic, and hemicellulolytic enzymes that were essential for the effective biodegradation of PKC. The substantial growth of selected L. plamtraum strains on PKC during SSF revealed the promising application of selected L. plantarum strains as a biotransformation agent for cellulosic biomass.

Optimized medium via statistical approach enhanced threonine production by Pediococcus pentosaceus TL-3 isolated from Malaysian food.

BACKGROUND: Threonine is an essential amino acid that is extensively used in livestock industry as feed supplement due to its pronounced effect in improving the growth performance of animals. Application of genetically engineered bacteria for amino acid production has its share of controversies after eosinophils myalgia syndrome outbreak in 1980s. This has urged for continuous search for a food grade producer as a safer alternative for industrial amino acid production. Lactic acid bacteria (LAB) appear as an exceptional candidate owing to their non-pathogenic nature and reputation of Generally Recognized as Safe (GRAS) status. Recently, we have identified a LAB, Pediococcus pentosaceus TL-3, isolated from Malaysian food as a potential threonine producer. Thus, the objective of this study was to enhance the threonine production by P. pentosaceus TL-3 via optimized medium developed by using Plackett-Burman design (PBD) and central composite design (CCD). RESULTS: Molasses, meat extract, (NH4)2SO4, and MnSO4 were identified as the main medium components for threonine production by P. pentosaceus TL-3. The optimum concentration of molasses, meat extract, (NH4)2SO4 and MnSO4 were found to be 30.79 g/L, 25.30 g/L, 8.59 g/L, and 0.098 g/L respectively based on model obtained in CCD with a predicted net threonine production of 123.07 mg/L. The net threonine production by P. pentosaceus TL-3 in the optimized medium was enhanced approximately 2 folds compared to the control. CONCLUSIONS: This study has revealed the potential of P. pentosaceus TL-3 as a safer alternative to produce threonine. Additionally, the current study has identified the key medium components affecting the production of threonine by P. pentosaceus TL-3, followed by optimization of their concentrations by means of statistical approach. The findings of this study could act as a guideline for the future exploration of amino acid production by LAB.

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.

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine).

BACKGROUND: The current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated. RESULTS: Expression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days). CONCLUSION: The presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

Extracellular Proteolytic Activity and Amino Acid Production by Lactic Acid Bacteria Isolated from Malaysian Foods.

Amino acids (AAs) are vital elements for growth, reproduction, and maintenance of organisms. Current technology uses genetically engineered microorganisms for AAs production, which has urged the search for a safer food-grade AA producer strain. The extracellular proteolytic activities of lactic acid bacteria (LAB) can be a vital tool to hydrolyze extracellular protein molecules into free AAs, thereby exhibiting great potential for functional AA production. In this study, eight LAB isolated from Malaysian foods were determined for their extracellular proteolytic activities and their capability of producing AAs. All studied LAB exhibited versatile extracellular proteolytic activities from acidic to alkaline pH conditions. In comparison, Pediococcus pentosaceus UP-2 exhibited the highest ability to produce 15 AAs extracellularly, including aspartate, lysine, methionine, threonine, isoleucine, glutamate, proline, alanine, valine, leucine, tryptophan, tyrosine, serine, glycine, and cystine, followed by Pediococcus pentosaceus UL-2, Pediococcus acidilactici UB-6, and Pediococcus acidilactici UP-1 with 11 to 12 different AAs production detected extracellularly. Pediococcus pentosaceus UL-6 demonstrated the highest increment of proline production at 24 h of incubation. However, Pediococcus acidilactici UL-3 and Lactobacillus plantarum I-UL4 exhibited the greatest requirement for AA. The results of this study showed that different LAB possess different extracellular proteolytic activities and potentials as extracellular AA producers.

Enhanced secretion of cyclodextrin glucanotransferase (CGTase) by Lactococcus lactis using heterologous signal peptides and optimization of cultivation conditions.

In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice.

BACKGROUND: Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world's population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0-80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed. RESULTS: A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice's spleen, lung and GIT. CONCLUSIONS: This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.