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Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) Lactococcus lactis is often reported to be insufficiently low due to limitations such as poor targeting and translocation by the signal peptide or degradation by the host proteases. In this study, the secretion efficiency in the host was enhanced through the utilization of a heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. The SPK1 was subjected to site-directed mutations targeting its tripartite N-, H-, and C-domains, and the effect on secretion efficiency as compared to the wild-type SPK1 and native lactococcal USP45 was determined on a reporter nuclease (NUC) of Staphylococcus aureus. A Fluorescence Resonance Energy Transfer (FRET) analysis indicated that four out of eight SPK1 variants successfully enhanced the secretion of NUC, with the best mutant, SPKM19, showing elevated secretion efficiency up to 88% (or by 1.4-fold) and an improved secretion activity yield of 0.292 ± 0.122 U/mL (or by 1.7-fold) compared to the wild-type SPK1. Modifications of the SPK1 at the cleavage site C-domain region had successfully augmented the secretion efficiency. Meanwhile, mutations in the H-domain region had resulted in a detrimental effect on the NUC secretion. The development of heterologous SPs with better efficacy than the USP45 has been demonstrated in this study for enhanced secretion of heterologous production and mucosal delivery applications in the lactococcal host.

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.

There is an increasing demand for natural food preservatives due to consumers' concern on the negative effects of chemical preservatives in food products. Nisin (bacteriocin) is an effective food biopreservative that has been approved globally. However, its low yield proves to be a limiting factor and must be addressed to meet the increasingly high demand from the food industry. The present work thus investigated the effects of individual and combined fermentation factors on Lactococcus lactis ATCC 11454 growth and nisin activity using the one-factor-at-a-time (OFAT) method. The level of each factor that gave the highest nisin production was then selected and combined to further improve its activity. The best combined conditions for highest cell growth and nisin activity were 30 °C, pH 6.0, and mild agitation with the addition of 1.0% w/v glucose, 1.0% w/v skim milk, and 0.5% v/v Tween 20. This increased nisin production by 22.7% as compared to control (basic condition). The present work provided critical information on the relationship between fermentation conditions, growth, and nisin activity of L. lactis ATCC 11454 that could be explored to understand the potential and limitation of the strain. This fermentation strategy can also serve as a benchmark to further enhance the production of bacteriocin or other biopreservative compounds.

The rise in in the number of drug-resistant bacteria that can resist almost all kinds of antibiotics is due to the overuse of these antibiotics (e.g., carbapenems). Thus, there is a need to find an alternative to antibiotic treatment such as the use of phages. In this study, phage UPM1705 was isolated from a polluted lake which can lyse its host Klebsiella pneumoniae ATCC BAA-1705. Based on morphological appearance from transmission electron microscopy, UPM1705 belongs to Caudovirales (Myoviridae). UPM1705 can reach a titer of 107 PFU/ml based on the double-layer method. It has a burst size of 298 PFU/bacteria cell and a latent period of 80 min, a rise period of 75 min, and adsorption time of 20 min based on a one-step growth curve assay using an MOI of 0.02. It was stable from 4°C to 80°C and retained its functionality at pH between 4 to 11, with pH of 7 being the optimum pH for the phage growth. The efficiency of UPM1705 was tested via a turbidity assay at MOI of 0.02, 0.2, and 2. UPM1705 was able to clear the turbidity of the host bacteria culture at all of these three MOIs. Thus, UPM1705 has the potential to be used for phage therapy.

Background. KRAS mutations are highly prevalent in pancreatic, lung, and colorectal carcinomas with G12V point substitution being one of the most prevalent mutations. While developments of peptide vaccines for KRAS(+) cancers are usually associated with poor immunogenicity, coupling mutant K-ras vaccines with universal CD4+ carrier molecules may enhance its outcome. Additionally, recent immunotherapeutic advances also suggest the possibility of inducing mucosal immunity against cancers using Lactococcus lactis as a live gastrointestinal delivery vehicle. Methods. A region of wild-type K-ras peptide was previously modified with a V7D substitution flanking the G12V mutation, generating a K-ras peptide (termed 68-V) with improved predicted antigenicity. This peptide was fused with a diphtheria toxoid sequence, and cloned into pNZ8048 vector within Lactococcus lactis NZ9000. BALB/c mice were then immunized orally, and then subjected to T/B cells immunophenotyping, as well as IgG and IgA detection. Results. Modified 68-V K-ras peptide and controls were successfully cloned and detection of His-tagged proteins expressed following induction by nisin was observed. Populations of CD3-CD19+ immune cells increased following immunization of 68-V, while K-ras specific-IgG and -IgA sera titers were elevated compared to wild-type and G12V K-ras controls. Conclusion. 68-V K-ras mimotope was shown to induce humoral-mediated immunity, highlighting the ability of an additional mutation flanking the G12V KRAS mutation to induce B cell activation and production of K-ras specific antibodies, while diphtheria toxoid was unable to stimulate an enhanced response when fused to 68-V. Nevertheless, these findings showed that further assessments are required to understand the role of K-ras specific antibodies within a KRAS(+) environment.

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.

Aims: To characterize bacteriophages with strong in vitro lytic activity against vancomycin resistant Enterococcus faecium before testing on the chicken skin for their efficacy. Study Design: An experimental was carried out to characterize two isolated bacteriophages against Enterococcus faecium and test for their efficacy on chicken skin. Study Place: The study was carried out in Laboratory of Vaccine and Immunotherapeutics, Institue of Bioscience, Universiti Putra Malaysia in Selangor, which is the most populous state in Malaysia. Methodology: Two host specific lytic phages against vancomycin resistant Enterococcus faecium strain FM8, designated as FM8-P1 and FM8-P2 were physiological characterized. This includes determination of their adsorption rate, multiplicity of infection, and single step growth kinetics. The optimum pH and temperature for both bacteriophages activity were also determined before tested Original Research Article Azlan et al.; AFSJ, 10(1): 1-14, 2019; Article no.AFSJ.49654 2 on chicken skin at 4°C and 25°C, which represent chiller and room temperature in poultry production line. Results: Based on the result of single-step growth kinetics, the latent period of FM8-P1 was 35 min with a burst size of 460 particles per infected cells, while FM8-P2 has a shorter latent period (20 min) but a smaller burst size of 60 particles. The highest adsorption rate for FM8-P1 was 83% and FM8-P2 was 90% at 2 min and 4 min respectively. Both bacteriophages also exihibited a wide range of pH and temperature for their activity. Conclusion: The specificity, lytic activity and stability of FM8-P1 and FM8-P2 emphasized their potential in effectively eliminating the vancomycin resistant Enterococcus faecium strain FM8. However, further works are required to validate their in situ reliability.

Microorganisms are the great sources of Natural Products (NPs); these are imperative to their survival apart from conferring competitiveness amongst each other within their environmental niches. Primary and secondary metabolites are the two major classes of NPs that help in cell development, where antimicrobial activity is closely linked with secondary metabolites. To capitalize on the effects of secondary metabolites, co-culture methods have been often used to develop an artificial microbial community that promotes the action of these metabolites. Different analytical techniques will subsequently be employed based on the metabolite specificity and sensitivity to further enhance the metabolite induction. Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS are commonly used for metabolite separation while Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) have been used as tools to elucidate the structure of compounds. This review intends to discuss current systems in use for co-culture in addition to its advantages, with discourse into the investigation of specific techniques in use for the detailed study of secondary metabolites. Further advancements and focus on co-culture technologies are required to fully realize the massive potential in synthetic biological systems.

KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses.

Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model.

The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.

Ingested dietary fibres are hydrolysed by colon microbiota to produce energy-providing short-chain fatty acids (SCFA) that stimulate anti-inflammatory effects. SCFA-producing bacteria were screened from bacteria isolated from human faeces using bromothymol blue as an acid indicator and gas chromatography for SCFA profiling. The beneficial functions (antagonistic activity, haemolytic activities, antibiotic susceptibility, mucus adherent percentage and toxin gene detection) were evaluated for the top five SCFA-producing bacteria isolated from three healthy volunteers that identified as Escherichia coli strains. They produced acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acids at average concentrations of 15.9, 1.8, 1.1, 1.9, 1.8, 2.7 and 3.4 mM, respectively. The SCFA production by E. coli strains was rapidly increased during the first 8 h of incubation and gradually decreased after 16 h of incubation. All E. coli strains showed acid and bile tolerance, resulting in a survival rate greater than 70% with no haemolytic activity, mucus adherence greater than 40% and susceptibility to conventional antibiotics. Hence, the selected E. coli strains exhibited promising probiotic properties with neither enterotoxin nor LPS producibility was detected. The present results confirm the existence of friendly and harmless E. coli strains in human microbiota as potential probiotics.

Background Extracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study. Results Escherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression. Conclusion SCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.

Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.

Vanda ‘Tan Chay Yan’, a beautiful golden-orange orchid with mild fragrance, is valuable in the floral industry. In this study, the volatile compounds from the flowers were profiled and a transcriptome library was created to search for fragrance-related transcripts. Volatiles from the tepals were captured using a SPME device with PDMS-coated fibre for GC-MS analysis. A transcriptome library was constructed and sequenced using the Illumina MiSeq Desktop Sequencer, de novo assembled using CLC Genomic Workbench version 6.0. BLASTx analysis, functional annotations and gene mapping of the annotated genes to the KEGG database were carried out using the Blast2go Pro software. Twelve compounds were identified in the volatiles including monoterpenes (93.19%), sesquiterpenes (4.32%) and traces of other compounds (2.49%) whereby cis-β-ocimene topped the list as the major constituent (88.41%). De novo assembly of the transcriptome generated a total of 34,027 sequences from which 20 fragrance-related transcripts in the terpenoid and benzenoid/phenylpropanoid biosynthetic pathways were identified. Combined information garnered from the volatiles and fragrance-related transcripts highlights the basic information on the fragrance constituents and biosynthetic pathways of this orchid which are useful for orchid breeders in selection of parents for new hybrids with fragrance as their value-added characteristic for the floriculture industry.

The effect of two signal peptides, namely Usp45 and Spk1 on the secretion of xylanase in Lactococcus lactis was analysed. 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. 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.

Aim To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice. Methods and results Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group. Conclusions Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect. Significance and impact of the study Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine.

The evolution of antimicrobial resistance (AMR) in pathogens has prompted extensive research to find alternative therapeutics. Plants rich with natural secondary metabolites are one of the go-to reservoirs for discovery of potential resources to alleviate this problem. Terpenes and their derivatives comprising of hydrocarbons, are usually found in essential oils (EOs). They have been reported to have potent antimicrobial activity, exhibiting bacteriostatic and bactericidal effects against tested pathogens. This brief review discusses the activity of terpenes and derivatives against pathogenic bacteria, describing the potential of the activity against AMR followed by the possible mechanism exerted by each terpene class. Finally, ongoing research and possible improvisation to the usage of terpenes and terpenoids in therapeutic practice against AMR are discussed.

This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 μg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.

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. Electronic supplementary material The online version of this article (10.1186/s12896-018-0461-y) contains supplementary material, which is available to authorized users.

Background Somatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions. Methods In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA. Results In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls. Discussion In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.

Background Somatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions. Methods In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA. Results In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls. Discussion In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.

This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 μg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.

Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis) has been gaining more interests for its positive prospects in genetic engineering-related applications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be able to express multiple genes in the eukaryotic system. Therefore, a cassette containing an internal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infectious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene, green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048 harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicistronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation system followed by SDS–PAGE and Western blot analysis to rapidly verify its functionality. Immunoblotting profiles showed the presence of 49 and 29 kDa bands that corresponds to the sizes of the VP2 and GFP proteins respectively. This preliminary result shows that the newly constructed lactococcal bicistronic vector can co-express multiple genes in a eukaryotic system via the IRES element thus suggesting its feasibility to be used for transfection of in vitro cell cultures and vaccine delivery.

Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile.

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

Background Bacterial biofilms are a preferred mode of growth for many types of microorganisms in their natural environments. The ability of pathogens to integrate within a biofilm is pivotal to their survival. The possibility of biofilm formation in Lactobacillus communities is also important in various industrial and medical settings. Lactobacilli can eliminate the colonization of different pathogenic microorganisms. Alternatively, new opportunities are now arising with the rapidly expanding potential of lactic acid bacteria biofilms as bio-control agents against food-borne pathogens. Results A new isolate Lactobacillus plantarum PA21 could form a strong biofilm in pure culture and in combination with several pathogenic and food-spoilage bacteria such as Salmonella enterica, Bacillus cereus, Pseudomonas fluorescens, and Aeromonas hydrophila. Exposure to Lb. plantarum PA21 significantly reduced the number of P. fluorescens,A. hydrophila and B. cereus cells in the biofilm over 2-, 4- and 6-day time periods. However, despite the reduction in S. enterica cells, this pathogen showed greater resistance in the presence of PA21 developed biofilm, either in the planktonic or biofilm phase. Lb. plantarum PA21 was also found to be able to constitutively express GFP when transformed with the expression vector pMG36e which harbors the gfp gene as a reporter demonstrating that the newly isolated strain can be used as host for genetic engineering. Conclusion In this study, we evaluate the ability of a new Lactobacillus isolate to form strong biofilm, which would provide the inhibitory effect against several spoilage and pathogenic bacteria. This new isolate has the potential to serve as a safe and effective cell factory for recombinant proteins.