Mu insertion in feuD triggers the increase in nisin immunity in Lactococcus lactis subsp. lactis N8.

AIMS: This study aims to explore how feuD mutation triggered the increase in nisin immunity of Lactococcus lactis L58, which was proven to be a feuD::Em-Mu mutant of Lc. lactis N8. METHODS AND RESULTS: The significant difference genes of Lc. lactis L58 and Lc. lactis N8 were compared at transcription and protein levels. Analysis revealed that the feuD mutation induced decrease in histidine-containing phosphocarrier protein PtsH (HPr) and increase in thioredoxin reductase TrxB (TR). Determination of iron concentration and cytoplasmic membrane potential (MP) showed the iron concentration decreased around 10% and the MP decreased approx. 14% in Lc. lactis L58. CONCLUSIONS: The increase in nisin immunity was dominated by TR up-expression by two main mechanisms in Lc. lactis L58. First, the TR-TRX (thioredoxin reductase) system changed the composition of cytoplasmic membrane by regulating the lipid metabolism to enhance the cells' resistance to nisin. Second, iron starvation stress induced decrease in MP; hence, the binding affinity of nisin to lipid II of Lc. lactis L58 decreased, which, in turn, increased the nisin immunity. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge on regulation mechanism of nisin immunity was enriched, and the theoretical basis for improving nisin production in engineering strain could be provided.

Lantibiotic immunity: inhibition of nisin mediated pore formation by NisI.

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC50 of 73 ± 10 nM, an 8-10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.

Lantibiotics: how do producers become self-protected?

Lantibiotics are small peptides produced by Gram-positive bacteria, which are ribosomally synthesized as a prepeptide. Their genes are highly organized in operons containing all the genes required for maturation, transport, immunity and synthesis. The best-characterized lantibiotic is nisin from Lactococcus lactis. Nisin is active against other Gram-positive bacteria via various modes of actions. To prevent activity against its producer strain, an autoimmunity system has developed consisting of different proteins, the ABC transporter NisFEG and a membrane anchored protein NisI. Together, they circumvent the ability of nisin to fulfill its action and cause cell death of L. lactis. Within this review, the mechanism of regulation, biosynthesis and activity of the immunity machinery will be discussed. Furthermore a short description about the application of these immunity proteins in both medical and industrial fields is highlighted.

Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains.

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.

Toll-like receptors and cytokines as surrogate biomarkers for evaluating vaginal immune response following microbicide administration.

Topical microbicides are intended for frequent use by women in reproductive age. Hence, it is essential to evaluate their impact on mucosal immune function in the vagina. In the present study, we evaluated nisin, a naturally occurring antimicrobial peptide (AMP), for its efficacy as an intravaginal microbicide. Its effect on the vaginal immune function was determined by localizing Toll-like receptors (TLRs-3, 9) and cytokines (IL-4, 6 , 10 and TNF-alpha) in the rabbit cervicovaginal epithelium following intravaginal administration of high dose of nisin gel for 14 consecutive days. The results revealed no alteration in the expression of TLRs and cytokines at both protein and mRNA levels. However, in SDS gel-treated group, the levels were significantly upregulated with the induction of NF-kappaB signalling cascade. Thus, TLRs and cytokines appear as sensitive indicators for screening immunotoxic potential of candidate microbicides.

Evaluation of developmental toxicity of microbicide Nisin in rats.

In the present study, we have investigated the developmental toxicity of a naturally occurring peptide, Nisin in rats in order to determine its suitability as a safe vaginal microbicide. Our earlier studies indicated that, Nisin is a dual function microbicide having contraceptive and antibacterial activities. However, as part of the safety evaluation of any vaginal microbicide, it is essential to determine its teratogenic potential in a suitable animal model before it is found suitable to enter clinical trials. Sixty pregnant rats allocated into four groups were orally administered with 10, 25 and 50 mg Nisin/kg/day from day 6 to day 15 of gestation. Individual food/water consumption and body weight changes were measured daily. Nisin did not cause maternal mortality nor did the treated animals show any clinical signs of toxicity when compared to the control animals. There were no biologically significant differences in maternal liver, kidney, thymus, ovary, gravid and empty uterine weights. Mean number of corpora lutea and implantation sites also did not differ in the treated groups when compared to their respective controls. All the fetuses were weighed, sexed and examined carefully for externally visible malformations. No gross external fetal alterations were observed at any dose tested. When stained by the double staining method, no skeletal malformations and visceral defects were observed in the fetuses. The growth and reproductive performance of the F1 progeny was also unaffected. In conclusion, Nisin shows unique clinical potential as a safe prophylactic microbicide to curb the transmission of STIs/HIV and unintended pregnancies.

Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin.

This review discusses the state-of-the-art in molecular research on the most prominent and widely applied lantibiotic, i.e., nisin. The developments in understanding its complex biosynthesis and mode of action are highlighted. Moreover, novel applications arising from engineering either nisin itself, or from the construction of totally novel dehydrated and/or lanthionine-containing peptides with desired bioactivities are described. Several challenges still exist in understanding the immunity system and the unique multiple reactions occurring on a single substrate molecule, carried out by the dehydratase NisB and the cyclization enzyme NisC. The recent elucidation of the 3-D structure of NisC forms the exciting beginning of further 3-D-structure determinations of the other biosynthetic enzymes, transporters and immunity proteins. Advances in achieving in vitro activities of lanthionine-forming enzymes will greatly enhance our understanding of the molecular characteristics of the biosynthesis process, opening up new avenues for developing unique and novel biocatalytic processes.

A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1.

Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with NukFEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG.

Distribution of the NisI immunity protein and enhancement of nisin activity by the lipid-free NisI.

Lactococcus lactis cells producing the antibacterial peptide nisin protect their own cytoplasmic membrane by specific immunity proteins, NisI and NisF/E/G. We show here that approximately half of the produced NisI escaped the lipid modification (LF-NisI=lipid-free NisI) and was secreted to the medium, and that LF-NisI had no affinity to cells of L. lactis. The molar ratio of NisI and nisin was determined to be approximately 1:10 on the cell surface and 1:50 in the culture supernatant. Purified LF-NisI was shown to enhance the activity of nisin against several tested indicator strains. The enhancement of nisin activity by LF-NisI was not observed with cells containing the NisFEG transport system.

Immunomodulatory effects of nisin in turbot (Scophthalmus maximus L.).

In the present work, the effect of nisin on the non-specific immune response of turbot (Scophthalmus maximus L.) leukocytes has been studied both in vitro and in vivo. The head kidney macrophage chemiluminescent (CL) response was significantly increased with intermediate doses of nisin (2.5 and 0.025 micro g ml(-1)) whilst the higher dose (25 micro g ml(-1)) significantly decreased the response after 24h incubation. When the incubation time was extended to 72 h, significant differences between doses were observed and the lower nisin concentration (0.025 micro g ml(-1)) appeared to be the optimum dose for increasing the CL response. The phagocytic activity of HK macrophages was also affected by in vitro nisin treatments. Nisin at 0.25 micro g ml(-1) and 0.025 micro g ml(-1) significantly stimulated the response after 24 and 72 h incubation respectively. Nitric oxide (NO) production by HK macrophages was not influenced by any nisin concentration employed for 24 or 72 h incubationsIn vivo, one week post injection, a slightly but non-significant stimulation of the CL response was observed with the lowest nisin concentration (0.0025 micro g fish(-1)). NO in serum and serum antibacterial index were not significantly affected by nisin treatments. On the other hand, lysozyme concentration in serum was significantly augmented with the lowest nisin dose (0.0025 micro g fish(-1)). The antibacterial effect of nisin against the fish pathogenic bacteria Carnobacterium piscicola (CECT 4020) was also demonstrated in vitro.

Antibacterial activities of nisin Z encapsulated in liposomes or produced in situ by mixed culture during cheddar cheese ripening.

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7 degrees C for 6 months. In some trials, L. innocua was added to cheese milk at 10(5) to 10(6) CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 +/- 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 +/- 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI.

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB 590, was constructed entirely of lactococcal DNA: the pSH 71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG 1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/ micro g DNA. MG 1614 carrying pLEB 590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB 590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.

Heterologous expression of the Lactococcus lactis bacteriocin, nisin, in a dairy Enterococcus strain.

The bacteriocin nisin is produced only by some strains of Lactococcus lactis, and to date production in other lactic acid bacteria has not been achieved. Enterococcus sp. strain N12beta is a nisin-immune transconjugant obtained from a nisin-producing donor (L. lactis ATCC 11454) and a dairy recipient (Enterococcus sp. strain S12beta), but it does not produce nisin. In this study, using PCR amplification, we confirmed that the whole nisin operon is likely present in Enterococcus sp. strain N12beta. Northern hybridization of total RNA from strain N12beta with a nisA probe and the results of reverse transcriptase PCR showed the lack of nisA transcription in this strain. However, nisA transcription was partially restored in strain N12beta upon growth in the presence of exogenous nisin, and the nisA transcription signal was intensified after an increase in the external nisin level. Furthermore, bioassays showed that active nisin was produced in a dose-dependent fashion by strain N12beta following induction by exogenous nisin. These results indicated that expression of the nisin genes in Enterococcus sp. strain N12beta depended on autoinduction via signal transduction. However, the amount of external inducing signal required was significantly greater than the amount needed for autoinduction in L. lactis.

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay.

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 1 of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at -70 degrees C, indicated that the dehydroalanine residue at position 5 (Dha5), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects.

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.

Quantification of nisin in flow-injection immunoassay systems.

A monoclonal-antibody-based, sequential competitive-flow-injection immunoassay system in expanded-bed mode has been developed for the determination of nisin. The system allows the determination of nisin in the presence of suspended particles without any significant interference, illustrating its potential for on-line monitoring of fermentation processes or the analysis of food matrices. The dose response range of the system when operated in expanded-bed mode was 6-90 microM. The detection limit under packed-bed conditions was 3 microM. The results correlated well with the results from conventional ELISA in the analysis of samples of processed cheese. When milk samples, fermentation samples and buffer were spiked with nisin, the mean recoveries were 86% for milk samples, 96% for fermentation samples and 98% for buffer solution.

Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains.

Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp. lactis biovar diacetylactis UL 719. Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z. Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum. The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively. In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount. In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected. In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.

Evaluation of immunomodulatory effects of nisin-containing diets on mice.

The effect of nisin on the immune response of mice was studied. Nisin (in the form of the commercial preparation Nisaplin) was incorporated in the diet of experimental mice which were fed for 30, 75 or 100 days. Short-term administration of diets containing Nisaplin induced an increase of both CD4 and CD8 T-lymphocyte cell counts and also a decrease of B-lymphocyte counts. After prolonged diet administration, T-cell counts returned to control levels. Normal levels of B-lymphocytes were also reached after prolonged administration of the lower (but not the higher) Nisaplin concentration. The macrophage/monocyte fraction isolated from peripheral blood became significantly increased after long-term administration (100 days) of Nisaplin-containing diets in a concentration-dependent way. Although the number of peritoneal cells was not affected by the diets, the phagocytic activity of peritoneal cells decreased after prolonged administration of low (but not high) Nisaplin doses.

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence.

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1.5 10(-1) international units per ml (IU ml-1), corresponding to 0.003 microgram ml-1 of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0.155 microgram ml-1 (7.9 IU ml-1) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.

Lysis of Lactococcus lactis subsp. cremoris SK110 and its nisin-immune transconjugant in relation to flavor development in cheese.

To develop a nisin-producing cheese starter, Lactococcus lactis subsp. cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.