M cell–targeting strategy enhances systemic and mucosal immune responses induced by oral administration of nuclease-producing L. lactis

Efficient delivery of antigens to the gut-associated lymphoid tissue (GALT) is the most critical step for the induction of mucosal immunity by oral vaccines. As M cells are the main portal for luminal antigens into the GALT, the M cell-targeting of antigens affords a promising strategy toward the development of effective oral vaccines. Lactococcus lactis is a fascinating recombinant host for oral vaccines, as they survive and produce antigens in the gut and have a particularly safe profile for human use. In this study, we developed and evaluated an M cell–targeting oral immunization system using recombinant L. lactis strains. For the purpose, we generated an L. lactis strain that secretes a model antigen fused with the OmpH β1α1 domain of Yersinia enterocolitica, which has been shown to bind to a complement C5a receptor on the M cell surface. As the model antigen, Staphylococcus aureus nuclease was used for fusion, resulting in L. lactis–expressing Nuc-OmpH (LL/Nuc-OmpH). Ex vivo intestinal loop assays showed that the amount of Nuc-OmpH taken up into Peyer’s patches was more than that of the unfused nuclease (Nuc). In addition, oral administration of the recombinant L. lactis strains to mice demonstrated that LL/Nuc-OmpH-induced nuclease-specific fecal IgA and serum IgG titers were significantly higher than those induced by LL/Nuc. These results indicate that OmpH works as an M cell–targeting molecule when fused with antigens secreted from L. lactis and that the M cell–targeting strategy affords a promising platform for L. lactis–based mucosal immunization.

     

Down-regulation of SLC35C1 induces colon cancer through over-activating Wnt pathway

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well-established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine-tuned remains elusive. We found that SLC35C1, a GDP-fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β-catenin is increased. Down-regulation of SLC35C1 is also detected by real-time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β-catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C-myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over-activation of Wnt signalling in colorectal cancer.     

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu-positive tumor cells

Targeting of non‐phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non‐pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti‐HER2/neu affibody on the surface. After co‐incubation with tumor cells for 3 h, the anti‐HER2/neu affibody‐presenting E. coli strain was selectively internalized into HER2/neu‐positive SKBR‐3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E‐mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor‐targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu‐positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier. Biotechnol. Bioeng. 2011; 108:1662–1672. © 2011 Wiley Periodicals, Inc.     

Serum antibodies to EpCAM in healthy donors but not ulcerative colitis patients

Purpose: The gastrointestinal carcinoma-associated antigen epithelial cell adhesion molecule (EpCAM) has been a target for passive and active immunotherapy of gastrointestinal carcinoma patients. The antigen is expressed by both tumor and normal tissues. The immunogenicity of EpCAM in colorectal cancer patients has been described previously. The purpose of this study was to evaluate humoral and cellular immune responses of healthy individuals and ulcerative colitis patients to EpCAM and to relate immune responses to colonic tissue expression of EpCAM. Methods: An inhibition radioimmunoassay was used to detect anti-EpCAM serum antibodies. Anti-EpCAM antibodies of a healthy donor were expressed by phages and sequenced. ³H-thymidine incorporation assay was used for detection of lymphoproliferative responses to stimulation with EpCAM. EpCAM tissue expression was determined by immunohistochemistry. Results: We detected anti-EpCAM serum antibodies in 4 of 10, and EpCAM-specific lymphoproliferation responses in 1 of 10 healthy volunteers. The majority of anti-EpCAM antibodies derived from a healthy donor were germline-encoded. In contrast, none of the 23 patients with ulcerative colitis showed serum antibodies to EpCAM (P=0.005). Antigen expression was greatly reduced and altered in ulcerative colitis patients, whereas colon from healthy individuals and uninvolved colon of colorectal cancer patients expressed high levels of EpCAM. Conclusion: The results of these studies suggest an association between EpCAM antibody production and colonic EpCAM expression in healthy individuals and patients with ulcerative colitis. Decreased and altered colonic EpCAM expression in ulcerative colitis patients may be related to the disease induction, based on the previously demonstrated adhesion function of this molecule. Healthy individuals with anti-EpCAM immune responses and high risk for developing colorectal carcinoma are prime candidates for prophylactic immunization against EpCAM.Emma E. Furth and Jian Li contributed equally     

Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non‐invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non‐invasive r‐ L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein‐expressing Caco‐2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

Effective suppression of tumour cells by oligoclonal HER2-targeted delivery of liposomal doxorubicin

Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (V_HH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.     

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.     

High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2^nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.     

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.     

Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer cells

A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the l-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z_HER2-BNC). For the investigation of binding affinity, Z_HER2-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z_HER2-BNC or Z_WT-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z_HER2-BNC mixed with liposomes was also compared with that of Z_WT-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z_HER2-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Expression of novel carotenoid biosynthesis genes from Enterococcus gilvus improves the multistress tolerance of Lactococcus lactis

Aims

To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid‐producing engineered MG1363 strain under stress condition was investigated.

Methods and Results

We cloned carotenoid biosynthesis genes from yellow‐pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid‐producing L. lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C₃₀ carotenoid 4,4′‐diaponeurosporene. After exposure to 32 mmol l^?1 H₂O₂, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml^?1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7‐, 6.8‐, 8.8‐ and 4.4‐fold higher, respectively, than those of MG1363 strain harbouring the empty vector pRH100.

Conclusions

The expression of carotenoid biosynthesis genes from Ent. gilvus improves the multistress tolerance of L. lactis.

Significance and Impact of the study

First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

miR‐150 functions as a tumour suppressor in human colorectal cancer by targeting c‐Myb

Our previously published study documented a deregulation of the microRNA miR‐150 in colorectal cancer. Here, we investigated further, in vitro and in vivo, the potential molecular mechanisms underlying the involvement of miR‐150 in colorectal cancer, using the appropriate molecular biological methods. We report that miR‐150 is a key regulator in the tumourigenesis and progression of colorectal cancer, by acting as a tumour suppressor targeting c‐Myb. The current findings suggest that miR‐150 may have important roles in the pathogenesis of colorectal cancer.     

A one step genotypic identification of Lactococcus lactis subspecies at the species/strain levels

A Lactococcus lactis subspecies-specific primer was designed based on their repetitive genome sequences. This primer enabled L. lactis subspecies to be identified simultaneously at both the species level and also the strain level. Based on studies using 70 strains of L. lactis and 60 strains of other non-target bacteria, the identification completely matched that obtained by the sequence of the 16S rRNA gene. However, inconsistency between phenotypic and genotypic characteristics was observed in some strains isolated from milk.