Enhancing activity of N-glycosylation for constitutive proteins secretions in non-polarized cells

Several fusion proteins of mouse Interleukins (mILs) and the enhanced green fluorescent protein (EGFP) were expressed in fibroblast and epithelial cells. Among these proteins, the mIL-31 derivative was the most efficiently secreted into the medium in a N-glycosylation-dependent manner. From the analysis of deletion mutants, the minimal structure for constitutive secretions consisted of a signal peptide and N-glycosylation. Introduction of the signal sequence from mIL-31 to human p53 protein failed to secrete the products, but further addition of the N-glycosylation site resulted in constitutive secretion of biologically active p53 protein into the medium in the N-glycosylated form. In this report, we showed the importance of N-glycosylation for constitutive protein secretions, especially using non-polarized cells.     

In situ delivery of cytokines by genetically engineered Lactococcus lactis

The development of novel approaches that allow for accurate targeting of therapeutics to the bowel mucosa is a priority in the research on inflammatory bowel disease. We have engineered Lactococcus lactis to secrete soluble, fully active, correctly processed cytokines. We have used these live, recombinant strains for the in situ delivery of mouse interleukin (mIL)-2, -6 and -10 at airway mucosa or mucosa of the colon. Strains that secrete mIL-2 or mIL-6 and produce TTFC intracellular show a higher level of anti-TTFC induction in mice following intranasal inoculation. We showed that mIL-10 producing L. lactis can prevent and cure enterocolitis in mice. The daily ingestion of this strain leads to the prevention of colitis in IL-10 –/– 129 Sv/Ev mice. The repeated addition of DSS to the drinking water of Balb/c mice leads to the induction of chronic colitis with a typical mean histological score of five points. Subsequent daily treatment with 10⁸ IL-10 producing L. lactis reduced the inflammation to a score of approximately 1 in 40% of the treated mice, which is a status equal to that of healthy control mice. Most other animals from the treated group only showed minor patchy remnants of the inflammation. Killing of the IL-10 producing bacteria by UV irradiation immediately prior to inoculation abrogates this therapeutic effect. Therefore it can be attributed to the active in vivo delivery of IL-10. We have further documented this by demonstrating in situ de novo synthesis of IL-10 in the colon of IL-10 –/– mice.     

Murine interferon-gamma inducible protein-10 (IP-10) secreted by Lactococcus lactis chemo-attracts human CD3+ lymphocytes

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.     

Functional Display of a Heterologous Protein on the Surface of Lactococcus lactis by Means of the Cell Wall Anchor of Staphylococcus aureus Protein A

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.     

Enhanced Secretion of Biologically Active Murine Interleukin-12 by Lactococcus lactis

The novel signal peptide SLPmod was used for the secretion of murine interleukin-12 (mIL-12) by Lactococcus lactis. A >4-fold increase in secretion was observed when SLPmod was used instead of the Usp45-derived secretion signal. Oral delivery of this cytokine using the autoinducible host L. lactis FI5876 utilizing SLPmod resulted in a significant increase in mIL-12 plasma levels in mice.     

Heterologous gene expression of bovine plasmin in Lactococcus lactis

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active. Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997     

Heterologous expression of secreted biologically active human interleukin-10 in Bifidobacterium breve

Construction of Bifidobacterium breve capable of production of secreted biologically active human interleukin-10 (hIL-10) is described. ORF coding for full-length mature human interleukin-10 was cloned into a series of expression vectors. This resulted in generation of translational fusions between hIL-10 and signal peptides sequences derived from Bifidobacterium breve genes sec2, apuB and B. adolescentis gene amyB under the control of constitutively active bifidobacterial promoter. We have shown that fusion to amyB signal peptide resulted in highest expression level of hIL-10 at the mRNA and protein level. Secreted hIL-10 was highly unstable in bifidobacterial culture supernatants in standard growth conditions. However, incubation of stationary cultures in buffered tissue culture medium resulted in production of stable biologically active hIL-10, albeit in low amounts (1.9 ng/ml).     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

Nisin-controlled extracellular production of apidaecin in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee (Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals.     

Lysozyme expression in Lactococcus lactis

Summary Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.     

Bovine Rotavirus Nonstructural Protein 4 Produced by Lactococcus lactis Is Antigenic and Immunogenic

Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.     

Efficient secretion of a modified E7 protein from human papilloma virus type‐16 by Lactococcus lactis

Aims: To create and provide a strain of the food‐grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type‐16. Methods and Results: We cloned the coding sequence of a modified E7 (E7m) from the HPV‐16 in a plasmid regulated by the strong expression promoter p59. Secretion of the E7m was made by the signal peptide of the usp45 gene. The E7m was detected by Western blot in the cell‐free‐medium fraction, showing no degradation or aberrant forms. Conclusions: We constructed a strain of L. lactis able to secrete efficiently a HPV‐16 E7 modified protein with diminished transforming activity. Significance and Impact of the Study: Human papilloma virus infection is associated with more than 99% of cervical cancers. Immunotherapy targeting E7 to treat HPV‐associated cervical malignancies has been demonstrated to be highly efficient. However, native E7 maintains transforming activity. We present this new strain of a food‐grade bacterium able to efficiently secrete a HPV‐16 E7‐modified protein with diminished transforming activity. This new strain could be used as a live vaccine to deliver E7 at a mucosal level and generate antitumour immune responses against HPV‐associated tumours.     

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10.     

Functional expression of mouse insulin-like growth factor-I with food-grade vector in Lactococcus lactis NZ9000

Aims: To functionally express the recombinant mouse insulin‐like growth factor‐I (rtmIGF‐I) in Lactococcus lactis NZ9000 with a food‐grade vector. Methods and Results: The rtmIGF‐I encoding sequence was inserted into secreted food‐grade vector pLEB688 and transformed into L. lactis NZ9000. The expression of the recombinant protein rtmIGF‐I was confirmed by tricine‐SDS‐PAGE analysis and Western blot. The concentration of this recombinant protein was 3 mg l^?1 in the medium fraction. Further experiment demonstrated that the recombinant protein was biologically active and promoted NIH3T3 cell proliferation in a concentration‐dependent manner. Conclusions: The rtmIGF‐I was expressed in L. lactis and located into the medium fraction. The optimal final concentration which could promote NIH3T3 cell proliferation after incubation was 100 ng ml^?1. Significance and Impact of the Study: The rtmIGF‐I was functionally expressed in L. lactis NZ9000 with a food‐grade vector. Thus, the recombinant L. lactis NZ9000 could act as a host for the production of rtmIGF‐I for further study. The recombinant strain could serve as an IGF‐I delivery system.     

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

Aims: The gram‐positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results: A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one‐step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. Conclusions: The one‐step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. Significance and Impact of the Study: This work provides an efficient method for identification of lactococcal strains of industrial importance.     

Effects of Intranasal Administration of a Leptin-Secreting Lactococcus lactis Recombinant on Food Intake, Body Weight, and Immune Response of Mice

Leptin is an adipocyte-derived pleiotropic hormone that modulates a large number of physiological functions, including control of body weight and regulation of the immune system. In this work, we show that a recombinant strain of the food-grade lactic acid bacterium Lactococcus lactis (LL-lep) can produce and efficiently secrete human leptin. The secreted leptin is a fully biologically active hormone, as demonstrated by its capacity to stimulate a STAT3 reporter gene in HEK293 cells transfected with the Ob-Rb leptin receptor. The immunomodulatory activity of leptin-secreting L. lactis was evaluated in vivo by coexpression with the human papillomavirus type 16 E7 protein. In C57BL/6 mice immunized intranasally with a recombinant L. lactis strain coproducing leptin and E7 antigen, the adaptive immune response was significantly higher than in mice immunized with recombinant L. lactis producing only E7 antigen, demonstrating adjuvanticity of leptin. We then analyzed the effects of intranasally administered LL-lep in obese ob/ob mice. We observed that daily administration of LL-lep to these mice significantly reduced body weight gain and food intake. These results demonstrate that leptin can be produced and secreted in an active form by L. lactis and that leptin-producing L. lactis regulates in vivo antigen-specific immune responses, as well as body weight and food consumption.     

Cell surface display system for Lactococcus lactis: a novel development for oral vaccine

The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. We constructed a plasmid vector pSVac that harbors a 255-bp single-repeat sequence of the cell wall-binding protein region of the AcmA protein. The recombinant plasmid was transformed into Escherichia coli and expression of the gene fragment was driven by the T7 promoter of the plasmid. SDS-PAGE showed the presence of the putative AcmA fragment and this was confirmed by Western blot analysis. The protein was isolated and purified using a His-tag affinity column. When mixed with a culture of L. lactis MG1363, ELISA and immunofluorescence assays showed that the cell wall-binding fragment was anchored onto the outer surface of the bacteria. This indicated that the AcmA repeat unit retained the active site for binding onto the cell wall surface of the L. lactis cells. Stability assays showed that the fusion proteins (AcmA/A1, AcmA/A3) were stably docked onto the surface for at least 5 days. The AcmA fragment was also shown to be able to strongly bind onto the cell surface of naturally occurring lactococcal strains and Lactobacillus and, with less strength, the cell surface of Bacillus sphericus. The new system designed for cell surface display of recombinant proteins on L. lactis was evaluated for the expression and display of A1 and A3 regions of the VP1 protein of enterovirus 71 (EV71). The A1 and A3 regions of the VP1 protein of EV71 were cloned upstream to the cell wall-binding domains of AcmA protein and successfully expressed as AcmA/A1 and AcmA/A3. Whole-cell ELISA showed the successful display of VP1 protein epitopes of EV71 on the surface of L. lactis. The success of the anchoring system developed in this study for docking the A1 and A3 epitopes of VP1 onto the surface of L. lactis cells opens up the possibilities of peptide and protein display for not only Lactococcus but also for other gram-positive bacteria. This novel way of displaying epitopes on the cell surface of L. lactis and other related organisms should be very useful in the delivery of vaccines and other useful proteins.     

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only ~60% of total Nuc was present in a secreted form at ~5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to ~80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ~20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the α-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.     

Construction of a food-grade multiple-copy integration system for Lactococcus lactis

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori⁺) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori⁺ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10⁻² copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest. Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His₆)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His₆-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His₆-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling.