Inhibitory Effect of Lactococcus lactis subsp. lactis HFY14 on Diphenoxylate-Induced Constipation in Mice by Regulating the VIP-cAMP-PKA-AQP3 Signaling Pathway.

AIM: The naturally fermented yak yogurt of pastoralists in the Tibetan Plateau, China, because of its unique geographical environment and the unique lifestyle of Tibetan pastoralists, is very different from other kinds of sour milk, and the microorganisms it contains are special. Lactococcus lactis subsp. lactis HFY14 (LLSL-HFY14) is a new lactic acid bacterium isolated from naturally fermented yak yogurt. The purpose of this study was to study the inhibitory effect of the bacterium on constipation. METHODS: Constipation was induced in ICR mice with diphenoxylate, and the constipated mice were treated with LLSL-HFY14. The weight and feces of the mice were visually detected. Colonic tissues were observed on hematoxylin and eosin-stained sections. Serum indices were detected with kits. mRNA expression in the colon was determined by quantitative polymerase chain reaction assay. RESULTS: Constipation caused weight loss, the number of defecation granules, defecation weight, fecal water content decreased, and the first black stool excretion time increased. LLSL-HFY14 alleviated these symptoms, and the effects were similar to those of lactulose (drug). The pathological examination revealed that constipation caused pathological changes in the colon, and LLSL-HFY14 effectively alleviated the disease. LLSL-HFY14 increased serum levels of motilin, gastrin, endothelin, substance P, acetylcholinesterase, and vasoactive intestinal peptide (VIP) and decreased serum levels of somatostatin in constipated mice. In addition, LLSL-HFY14 upregulated VIP, cAMP, protein kinase A, and aquaporin 3 expression in colonic tissues of constipated mice in a dose-dependent manner. CONCLUSION: LLSL-HFY14 inhibited constipation, similar to lactulose, and has the potential to become a biological agent.

Probiotic Activity of Enterococcus faecium and Lactococcus lactis Isolated from Thai Fermented Sausages and Their Protective Effect Against Clostridium difficile.

Lactic acid bacteria, Enterococcus faecium and Lactococcus lactis, previously isolated from Thai fermented sausages were elucidated their probiotic properties especially in the control of Clostridium difficile 630. Both isolates survived in simulated gastric solution at pH 3 followed in simulated intestinal solution at pH 8. The presence of skimmed milk also helped the bacteria to survive through acidic and alkaline in gastrointestinal conditions. The adhesion properties of both isolates were tested using a human colon adenocarcinoma cell line. The result showed that both isolates exhibited desirable probiotic properties which adhered to Caco-2 cells. The neutralized cell-free supernatant of both isolates demonstrated that no cytotoxicity toward Caco-2 cells vice versa cell-free supernatant of C. difficile 630 toward Caco-2 cell demonstrated high toxicity. The immunomodulation effect in response to bacterial neutralized cell-free supernatant and cell-free supernatant was also studied. The expression level of pro-inflammatory cytokine of Caco-2 cell which are tumor necrosis factor-α and interleukin-8 was evaluated using quantitative reverse transcriptase PCR. Both isolates were able to diminish the expression level of TNF-α and IL-8 induced by the cell-free supernatant of C. difficile 630. Hence, these isolates would be able to improve the gut health through counteracting the C. difficile-associated intestinal inflammation in human cell lines. These results may contribute to the development of the isolates using as probiotics.

Antagonistic activity of recombinant Lactococcus lactis NZ1330 on the adhesion properties of Escherichia coli causing urinary tract infection.

Death from infectious diseases has caused concerns about increases in the resistance of pathogens, impelling researchers to create novel therapeutic solutions. The management of intestinal tract problems has been the advance use of probiotics in medicine. The aim of this study was evaluate the physicochemical cell surface and adhesion properties of recombinant Lacotococcus lactis NZ1330 containing Ama r 2 gene, followed by the assessment of the antagonistic activity of this strain against the Escherichia coli causing urinary tract infection (UTI) in humans. For this purpose, cloning and expression of Ama r 2 gene were done. Afterwards, acid and bile resistance, which are the primary characteristics of any probiotic, were evaluated. The r-L. lactis NZ1330 was examined for the physicochemical properties of cell surfaces and the adhesion properties against Escherichia coli. Furthermore, the potential of the recombinant strain to adhere to adenocarcinoma intestinal cell line, Caco-2 cells, as well as the antagonistic properties of r-L. lactis NZ1330 against E. coli was investigated. r-L. lactis NZ1330 was capable of surviving at low pH and different concentrations of bile salts. 40.1% hydrophobicity, 36.5% auto-aggregation and 14.4% co-aggregation were observed for this strain. The adhesion level of r-L. lactis NZ1330 was 5.7% which was also confirmed by scanning electron microscopy (SEM). r-L. lactis NZ1330 was able to compete, inhibit and displace the adhesion of Escherichia coli to Caco-2 cells. r-L. lactis NZ1330 was considered to be a reliable probiotic alternative by showing these desirable properties. Results revealed that Ama r 2 gene expression had no effect on the positive probiotic properties of L. lactis NZ1330, proving this strain could be a suitable probiotic host for the expression of this allergen.

Staphylococcus aureus Epicutaneous Infection Is Suppressed by Lactococcus lactis Strain Plasma via Interleukin 17A Elicitation.

BACKGROUND: Lactococcus lactis strain Plasma (LC-Plasma) was revealed to stimulate plasmacytoid dendritic cells and induce antiviral immunity in vitro and in vivo. In this study, we assessed the effects of LC-Plasma on skin immunity. METHODS: To evaluate the effect of LC-Plasma on skin immunity and Staphylococcus aureus epicutaneous infection, lymphocyte activities in skin-draining lymph nodes (SLNs) and gene expression in skin were analyzed after 2 weeks of oral administration of LC-Plasma. To evaluate the mechanisms of interleukin 17A production, SLN lymphocytes were cultured with or without LC-Plasma, and the interleukin 17A concentrations in supernatants were measured. RESULTS: Oral administration of LC-Plasma activated plasma dendritic cells in SLNs, augmented skin homeostasis, and elicited suppression of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes proliferation. In addition, significant suppression of the S. aureus burden and reduced skin inflammation were observed following oral administration of LC-Plasma. Furthermore, a subsequent in vitro study revealed that LC-Plasma could elicit interleukin 17A production from CD8+ T cells and that its induction mechanism depended on the Toll-like receptor 9 signaling pathway, with type I interferon partially involved. CONCLUSIONS: Our results suggest that LC-Plasma oral administration enhances skin homeostasis via plasma dendritic cell activation in SLNs, resulting in suppression of S. aureus epicutaneous infection and skin inflammation.

Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP.

Mucositis is an inflammatory condition of the gut, caused by an adverse effect of chemotherapy drugs, such as 5-fluorouracil (5-FU). In an attempt to develop alternative treatments for the disease, several research groups have proposed the use of probiotics, in particular, Lactic Acid Bacteria (LAB). In this context, the use of recombinant LAB, for delivering anti-inflammatory compounds has also been explored. In previous work, we demonstrated that either Lactococcus lactis NZ9000 or a recombinant strain expressing an antimicrobial peptide involved in human gut homeostasis, the Pancreatitis-associated Protein (PAP), could ameliorate 5-FU-induced mucositis in mice. However, the impact of these strains on the gut microbiota still needs to be elucidated. Therefore, in the present study, we aimed to characterize the effects of both Lactococci strains in the gut microbiome of mice through a 16 S rRNA gene sequencing metagenomic approach. Our data show 5-FU caused a significant decrease in protective bacteria and increase of several bacteria associated with pro-inflammatory traits. The Lactococci strains were shown to reduce several potential opportunistic microbes, while PAP delivery was able to suppress the growth of Enterobacteriaceae during inflammation. We conclude the strain secreting antimicrobial PAP was more effective in the control of 5-FU-dysbiosis.

A Mixture of Five Bacterial Strains Attenuates Skin Inflammation in Mice.

BACKGROUND: There is a growing interest in the effects of probiotics for the prevention and treatment of skin diseases due to their immunomodulatory and antiinflammatory properties. OBJECTIVE: To assess a mixture of five bacterial strains in the prevention of chronic skin inflammation in mice. METHODS: Hairless SKH-1 mice received daily oral treatment with the probiotic mixture at the dose of 1x109 Colony-Forming Unit (CFU)/day (or vehicle) for three weeks. Chronic skin inflammation was induced by repeated applications of 12-O-tetradecanoylphorbol-13- acetate (TPA; control mice received acetone). Macroscopic and microscopic evaluations of skin lesions were performed and serum levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, IL-17, IL-22, IL-10 and IL-4 measured at the end of the study. RESULTS: Treatment with the probiotic mixture significantly limited the induced chronic skin inflammation at both the macroscopic and microscopic levels. This limitation was consistent with downregulated levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, IL- 17 and IL-22) and up-regulated levels of the anti-inflammatory cytokines, IL-10 and IL-4. CONCLUSION: The results suggest that the probiotic mixture tested could help in preserving skin integrity and homeostasis and that its use could be beneficial in dermatological conditions such as atopic dermatitis and psoriasis.

Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

Effect of thyme essential oil and Lactococcus lactis CBM21 on the microbiota composition and quality of minimally processed lamb's lettuce.

The main aim of this work was to evaluate, at pilot scale in an industrial environment, the effects of the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil compared to chlorine, used in the washing step of fresh-cut lamb's lettuce, on the microbiota and its changes in relation to the time of storage. The modification of the microbial population was studied through pyrosequencing in addition to the traditional plate counts. In addition, the volatile molecule and sensory profiles were evaluated during the storage. The results showed no significant differences in terms of total aerobic mesophilic cell loads in relation to the washing solution adopted. However, the pyrosequencing data permitted to identify the genera and species able to dominate the spoilage associations over storage in relation to the treatment applied. Also, the analyses of the volatile molecule profiles of the samples during storage allowed the identification of specific molecules as markers of the spoilage for each different treatment. The sensory analyses after 3 and 5 days of storage showed the preference of the panelists for samples washed with the combination thyme EO and the biocontrol agent. These samples were preferred for attributes such as flavor, acceptability and overall quality. These results highlighted the effect of the innovative washing solutions on the quality of lettuce through the shift of microbiota towards genera and species with lower potential in decreasing the sensory properties of the product.

Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain.

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

Ectopic Expression of Innate Immune Protein, Lipocalin-2, in Lactococcus lactis Protects Against Gut and Environmental Stressors.

BACKGROUND: Lipocalin-2 (Lcn2) is a multifunctional innate immune protein that exhibits antimicrobial activity by the sequestration of bacterial siderophores, regulates iron homeostasis, and augments cellular tolerance to oxidative stress. Studies in the murine model of colitis have demonstrated that Lcn2 deficiency exacerbates colitogenesis; however, the therapeutic potential of Lcn2 supplementation has yet to be elucidated. In light of its potential mucoprotective functions, we, herein, investigated whether expression of Lcn2 in the probiotic bacterium can be exploited to alleviate experimental colitis. METHODS: Murine Lcn2 was cloned into the pT1NX plasmid and transformed into Lactococcus lactis to generate L. lactis-expressing Lcn2 (Lactis-Lcn2) or the empty plasmid (Lactis-Con). Lactis-Lcn2 was characterized by immunoblot and enzyme-linked immunosorbent assay and tested for its antimicrobial efficacy on Escherichia coli. The capacity of Lactis-Lcn2 and Lactis-Con to withstand adverse conditions was tested using in vitro viability assays. Dextran sodium sulfate colitis model was used to investigate the colonization ability and therapeutic potential of Lactis-Lcn2 and Lactis-Con. RESULTS: Lcn2 derived from Lactis-Lcn2 inhibited the growth of E. coli and reduced the bioactivity of enterobactin (E. coli-derived siderophore) in vitro. Lactis-Lcn2 displayed enhanced tolerance to adverse pH, high concentration of bile acids, and oxidative stress in vitro and survived better in the inflamed gut than Lactis-Con. Consistent with these features, Lactis-Lcn2 displayed better mucoprotection against intestinal inflammation than Lactis-Con when administered into mice with dextran sulfate sodium-induced acute colitis. CONCLUSIONS: Our findings suggest that Lcn2 expression can be exploited to enhance the survivability of probiotic bacteria during inflammation, which could further improve its efficacy to treat experimental colitis.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Anti-cancer effect of lactic acid bacteria expressing antioxidant enzymes or IL-10 in a colorectal cancer mouse model.

The association between inflammatory bowel diseases and colorectal cancer is well documented. The genetic modification of lactic acid bacteria as a tool to increase the anti-inflammatory potential of these microorganisms has also been demonstrated. Thus the aim of the present work was to evaluate the anti-cancer potential of different genetically modified lactic acid bacteria (GM-LAB) producing antioxidant enzymes (catalase or superoxide dismutase) or the anti-inflammatory cytokine IL-10 (protein or DNA delivery) using a chemical induced colon cancer murine model. Dimethilhydrazine was used to induce colorectal cancer in mice. The animals received GM-LAB producing anti-oxidant enzymes, IL-10 or a mixture of different GM-LAB. Intestinal damage, enzyme activities and cytokines were evaluated and compared to the results obtained from mice that received the wild type strains from which derived the GM-LAB. All the GM-LAB assayed showed beneficial effects against colon cancer even though they exerted different mechanisms of action. The importance to select LAB with innate beneficial properties as the progenitor strain was demonstrated with the GM-LAB producing anti-oxidant enzymes. In addition, the best effects for the mixtures GM-LAB that combine different anti-inflammatory mechanism. Results indicate that mixtures of selected LAB and GM-LAB could be used as an adjunct treatment to decrease the inflammatory harmful environment associated to colorectal cancer, especially for patients with chronic intestinal inflammation who have an increased risk to develop colorectal cancer.

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow.

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.

Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus.

Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated Lactococcus lactis V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for Escherichia coli and for Staphylococcus aureus, two major mastitis pathogens, we investigated L. lactis V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. L. lactis V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with E. coli was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that L. lactis V7 inhibited the internalisation of both E. coli and S. aureus strains into bMEC, whereas it inhibited the adhesion of only one out of the two S. aureus strains and of none of the E. coli strains tested. Investigation of the bMEC immune response showed that L. lactis V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the E. coli strains. Altogether these features of L. lactis V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer.

Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen ß-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.

Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines.

Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 µg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics.

Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (µ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at µ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at µ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712.

The temperate bacteriophage TP712 was unable to plaque on Lactococcus lactis ΔftsH lacking the membrane protease FtsH and complementation in trans restored the WT phenotype. Absence of ftsH did not hinder phage adsorption, phage DNA delivery or activation of the lytic cycle. Thin sections revealed that TP712 virions appeared to be correctly assembled inside the ΔftsH host, but were not released. These virions were infective, demonstrating that a functional host FtsH is required by TP712 to proceed effectively with lysis of the host.

Oxidative stress at high temperatures in Lactococcus lactis due to an insufficient supply of Riboflavin.

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen.