Immunomodulatory effects of different strains of Lactococcus lactis in DSS-induced colitis.

Inflammatory bowel diseases (IBD) are gastrointestinal disorders characterized by a breakdown in intestinal homeostasis by inflammatory immune responses to luminal antigens. Novel strategies for ameliorating IBD have been proposed in many studies using animal models. Our group has demonstrated that administration of Lactococcus lactis NCDO 2118 can improve clinical parameters of colitis induced by oral administration of dextran sulphate sodium (DSS). However, it is not clear whether other strains of L. lactis can yield the same effect. The objective of present study was to analyze the effects of three different L. lactis strains (NCDO2118, IL1403 and MG1363) in the development of DSS-induced colitis in C57BL/6 mice. Acute colitis was induced in C57/BL6 mice by the administration of 2% DSS during 7 consecutive days. Body weight loss and shortening of colon length were observed in DSS-treated mice, and none of L. lactis strains had an impact in these clinical signs of colitis. On the other hand, all strains improved the global macroscopical disease index and prevented goblet cells depletion as well as the increase of intestinal permeability. TNF-α production was reduced in gut mucosa of L. lactis DSS-treated mice indicating a modulation of a critical pro-inflammatory response by all strains tested. However, only L. lactis NCDO2118 and MG1363 induced a higher frequency of CD11c+CD11b-CD103+ tolerogenic dendritic cells in lymphoid organs of mice at steady state. We conclude that all tested strains of L. lactis improved the clinical scores and parameters of colitis, which confirm their anti-inflammatory properties in this model of colitis.

Lactococcus lactis FNBPA+ (pValac:e6ag85a) Induces Cellular and Humoral Immune Responses After Oral Immunization of Mice.

The development of a new vaccine strategy against tuberculosis is urgently needed and has been greatly encouraged by the scientific community worldwide. In this work, we constructed a lactococcal DNA vaccine based on the fusion of two Mycobacterium tuberculosis antigens, ESAT-6 and Ag85A, and examined its immunogenicity. The coding sequences of the ESAT-6 and Ag85A genes were fused and cloned into the eukaryotic expression pValac vector, and the functionality of the vector was confirmed in vitro. Then, L. lactis FnBPA+ (pValac:e6ag85a) was obtained and used for oral immunization of mice. This strain induced significant increases in IFN-γ, TNF-α, and IL-17 cytokines in stimulated splenocyte cultures, and significant production of antigen-specific sIgA was observed in the colonic tissues of immunized mice. We demonstrated that L. lactis FnBPA+ (pValac:e6ag85a) generated a cellular and humoral immune response after oral immunization of mice. The strategy developed in this work may represent an interesting DNA mucosal vaccine candidate against tuberculosis, using the fusion of two highly immunogenic antigens delivered by safe lactic acid bacteria.

Recombinant Lactococcus lactis Carrying IL-4 and IL-10 Coding Vectors Protects against Type 1 Diabetes in NOD Mice and Attenuates Insulitis in the STZ-Induced Model.

Type 1 diabetes (T1D) is an autoimmune disease that culminates in beta cell destruction in the pancreas and, subsequently, deficiency in insulin production. Cytokines play a crucial role in the development of diabetes, orchestrating the recruitment and action of immune cells, to not only destroy insulin-producing cells but also preserve them. Therefore, the aim of this study was to investigate the effect of orally administered Lactococcus lactis MG1363 FnBPA+ strains carrying plasmids encoding IL-4 and IL-10 in the streptozotocin- (STZ-) induced diabetes model and in nonobese diabetic (NOD) mice. The STZ-induced mice that were treated with combined bacterial strains carrying plasmids encoding IL-4 and IL-10 showed lower incidence of diabetes and more preserved pancreatic islets than the mice that received the individual bacterial strains. Combined administration of L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) and L. lactis MG1363 FnBPA+ (pValac::IL-10) resulted in protection against diabetes in NOD mice. It was shown that the combined treatment with recombinant bacterial by oral route prevented hyperglycemia and reduced the pancreatic islets-destruction in NOD mice. In addition, increased levels of IL-4 and IL-10 in serum and pancreatic tissue revealed a systemic effect of the treatment and also favored an anti-inflammatory microenvironment. Reduced concentrations of IL-12 in pancreas were essential to the regulation of inflammation, resulting in no incidence of diabetes in treated NOD mice. Normal levels of intestinal sIgA after long-term treatment with the L. lactis strains carrying plasmids encoding IL-4 and IL-10 indicate the development of oral tolerance and corroborate the use of this potent tool of mucosal delivery. For the first time, L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10 are tested in STZ-induced and NOD mouse models. Therefore, our study demonstrates this innovative strategy provides immunomodulatory potential for further investigations in T1D and other autoimmune diseases.

Mycobacterial Hsp65 antigen delivered by invasive Lactococcus lactis reduces intestinal inflammation and fibrosis in TNBS-induced chronic colitis model.

Intestinal fibrosis associated with Crohn's disease (CD), which a common and serious complication of inflammatory bowel diseases. In this context, heat shock proteins (HSPs) might serve as an alternative treatment because these antigens play important roles in the regulation of effector T cells. We thus evaluated the anti-inflammatory and antifibrotic capacities of an invasive and Hsp65-producing strain-Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65)-in chronic intestinal inflammation to assess its potential as an alternative therapeutic strategy against fibrotic CD. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in BALB/c mice, and the mice were treated orally with L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) via intragastric gavage. The oral administration of this strain significantly attenuated the severity of inflammation and intestinal fibrosis in mice (p < 0.05). These results are mainly justified by reductions in the levels of the pro-fibrotic cytokines IL-13 and TGF-ß and increases in the concentration of the regulatory cytokine IL-10. The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to reductions in the severity of inflammatory damage in chronic experimental CD, and these findings confirm the effectiveness of this new antifibrotic strategy based on the delivery of therapeutic proteins to inside cells of the host intestinal mucosa.

Hsp65-Producing Lactococcocus lactis Prevents Antigen-Induced Arthritis in Mice.

Oral tolerance is the physiological process that enables the immune system to differentiate between harmless dietary and microbiota antigens from pathogen derived antigens. It develops at the mucosal surfaces and can result in local and systemic regulatory and anti-inflammatory effects. Translation of these benefits to the clinical practice faces limitations involving specificity and doses of antigen as well as regimens of feeding. To circumvent these problems, we developed a recombinant Hsp65 delivered by the acid lactic bacteria Lactococcus lactis NCDO 2118 directy in the intestinal mucosa. Hsp65 is a ubiquitous protein overexpressed in inflamed tissues and capable of inducing immunoregulatory mechanisms. L. lactis has probiotic properties and is commonly and safely used in dairy products. In this study, we showed that continuous delivery of HSP65 in the gut mucosa by L. lactis is a potent tolerogenic stimulus inducing regulatory CD4+LAP+ T cells that prevented collagen-induced and methylated bovine serum albumin-induced arthritis in mice. Clinical and histological signs of arthritis were inhibited as well as levels of inflammatory cytokines such as IL-17 and IFN-γ, serum titers of anti-collagen antibodies and rheumatoid factor. Oral administration of L. lactis induced alterations in microbiota composition toward an increased abundance of anaerobic bacteria such as Bifidobacterium and Lactobacillus. Tolerance to HSP65 and arthritis prevention induced by the recombinant L. lactis was associated with increase in IL-10 production by B cells and it was dependent on LAP+ T cells, IL-10 and TLR2 signaling. Therefore, HSP65-producing treatment induced effective tolerance and prevented arthritis development suggesting it can be used as a therapeutic tool for autoimmune diseases.

Attenuation of intestinal inflammation in IL-10 deficient mice by a plasmid carrying Lactococcus lactis strain.

BACKGROUND: Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract (GIT) and to date, no efficient treatments exist. Interleukin-10 (IL-10), one of the most important anti-inflammatory cytokines of the immune response, has been under study due to its potential for IBD therapy; however, systemic treatments lead to undesirable side effects and oral administration is limited due to its quick degradation. To avoid these bottlenecks, we previously engineered an invasive Lactococcus lactis (L. lactis) strain capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) that diminished inflammation in two induced mouse models of intestinal inflammation. Thus, the aim of this study was to analyze its therapeutic effect in the IL-10-deficient mouse model (IL-10-/-) that spontaneously and gradually develops an inflammation that modifies the immune system and resembles Crohn's disease (CD) in humans, and evaluate if it would also diminish and/or prevent the onset of this disease. RESULTS: Oral administration of L. lactis MG1363 FnBPA+ (pValac:il-10) to IL-10-/- mice not only led to IL-10 production by these, but consequently also diminished the severe development of the disease, with animals showing lower macroscopic scores and histological damages, increased IL-10 levels and tendency to lower pro-inflammatory cytokine levels. CONCLUSIONS: The results of this study, together with the previously published ones using this DNA delivery-based strategy, show that it is capable of creating and maintaining an anti-inflammatory environment in the GIT and thus effectively diminish the onset of inflammation in various mouse models.

Mucosal delivery of Lactococcus lactis carrying an anti-TNF scFv expression vector ameliorates experimental colitis in mice.

BACKGROUND: Anti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice. RESULTS: Experimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1ß, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis. CONCLUSION: These results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.

A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways.

Heat shock proteins (Hsps) are highly expressed at all sites of inflammation. As they are ubiquitous and immunodominant antigens, these molecules represent good candidates for the therapeutic use of oral tolerance in autoimmune and chronic inflammatory diseases. Evidences from human and animal studies indicate that inflammatory bowel disease (IBD) results from uncontrolled inflammatory responses to intestinal microbiota. Hsps are immunodominant proteins expressed by several immune cells and by commensal bacteria. Using an IBD mouse model, we showed that oral pretreatment with genetically modified Lactococcus lactis that produces and releases Mycobacterium Hsp65, completely prevented DSS-induced colitis in C57BL/6 mice. Protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; increased IL-10 production in colonic tissue; and expansion of CD4+Foxp3+ and CD4+LAP+ regulatory T cells in spleen and mesenteric lymph nodes. This effect was dependent on IL-10 and toll-like receptor 2. Thus, this approach may open alternative options for long-term management of IBD.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

BACKGROUND: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. RESULTS: During this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. CONCLUSIONS: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells.

BACKGROUND: Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease. RESULTS: The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid-pValac::dts::IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C(-)IL-4(+), F4/80(+)MHCII(+)Ly6C(-)IL-10(+), F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups. CONCLUSIONS: This study shows that L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels of IL-10-secreting regulatory cells and, thus, demonstrating the effectiveness of this novel DNA delivery-based strategy.

Expression of fibronectin binding protein A (FnBPA) from Staphylococcus aureus at the cell surface of Lactococcus lactis improves its immunomodulatory properties when used as protein delivery vector.

A recombinant strain of Lactococcus lactis displaying a cell-surface anchored fibronectin binding protein A (FnBPA) from Staphylococcus aureus (LL-FnBPA) had been shown to be more efficient in delivering plasmid than its wild-type counterpart both in vitro and in vivo, and have the ability to orientate the immune response toward a Th2 profile in a context of a DNA vaccination. The aim of this work was to test whether this LL-FnBPA strain could shape the immune response after mucosal administration in mice. For this, we used a mouse model of human papilloma virus (HPV)-induced cancer and a L. lactis strain displaying at its cell surface both HPV-16-E7 antigen (LL-E7) and FnBPA (LL-E7+FnBPA). Our results revealed a more efficient systemic Th1 immune response with recombinant LL-E7+FnBPA. Furthermore, mice vaccinated with LL-E7+FnBPA were better protected when challenged with HPV-16-induced tumors. Altogether, the results suggest that FnBPA displays adjuvant properties when used in the context of mucosal delivery using L. lactis as a live vector.

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.

Previous Ingestion of Lactococcus lactis by Ethanol-Treated Mice Preserves Antigen Presentation Hierarchy in the Gut and Oral Tolerance Susceptibility.

BACKGROUND: Ethanol (EtOH) consumption is able to disturb the ovalbumin (OVA)-oral tolerance induction by interfering on the function of antigen presenting cells (APC), down-regulating dendritic cells (DCs) and macrophages and up-regulating B-lymphocytes and their function, which results in an overall allergic-type immune status. In this study, the potential of a priori administration of Lactococcus lactis (LL) in avoiding loss of oral tolerance in EtOH-treated mice was investigated. METHODS: Female C57BL/6 mice received, by oral route, ad libitum wild-type (WT) LL or heat-shock protein producer (Hsp65) LL for 4 consecutive days. Seven days later, mice were submitted to short-term high-dose EtOH treatment. After 24 hours, stomach, intestine, spleen, mesenteric lymph nodes (mLN) specimens were collected for biomarkers analysis. Following EtOH-treatment protocol, a group of animals underwent single-gavage OVA-tolerance protocol and sera samples collected for antibody analysis. RESULTS: The ingestion of WT LL or Hsp65 LL is able to restore oral tolerance to OVA in EtOH-treated mice, by reducing local and systemic allergic outcomes such as gastric mast cells and gut-interleukin-4, as well as serum IgE. WT LL treatment prevents the decrease of mLN regulatory T cells induced by the EtOH treatment. Moreover, LL treatment preserves APC hierarchy and antigen presentation commitment in EtOH-treated mice, with conserved DC and macrophage activity over B lymphocytes in mLN and preserved macrophage activity over DC and B-cell subsets in the spleen. CONCLUSIONS: The present findings suggest that a priori ingestion of LL preserves essential mechanisms associated with oral tolerance induction that are disturbed by EtOH ingestion. Maintenance of mucosal homeostasis by preserving APC hierarchy and antigen presentation commitment could be associated with T-regulatory subset activities in the gastrointestinal tract.

Current Review of Genetically Modified Lactic Acid Bacteria for the Prevention and Treatment of Colitis Using Murine Models.

Inflammatory Bowel Diseases (IBD) are disorders of the gastrointestinal tract characterized by recurrent inflammation that requires lifelong treatments. Probiotic microorganisms appear as an alternative for these patients; however, probiotic characteristics are strain dependent and each probiotic needs to be tested to understand the underlining mechanisms involved in their beneficial properties. Genetic modification of lactic acid bacteria (LAB) was also described as a tool for new IBD treatments. The first part of this review shows different genetically modified LAB (GM-LAB) described for IBD treatment since 2000. Then, the two principally studied strategies are discussed (i) GM-LAB producing antioxidant enzymes and (ii) GM-LAB producing the anti-inflammatory cytokine IL-10. Different delivery systems, including protein delivery and DNA delivery, will also be discussed. Studies show the efficacy of GM-LAB (using different expression systems) for the prevention and treatment of IBD, highlighting the importance of the bacterial strain selection (with anti-inflammatory innate properties) as a promising alternative. These microorganisms could be used in the near future for the development of therapeutic products with anti-inflammatory properties that can improve the quality of life of IBD patients.

Evaluation of a Streptococcus thermophilus strain with innate anti-inflammatory properties as a vehicle for IL-10 cDNA delivery in an acute colitis model.

The aim of this work was to develop a Streptococcus (S.) thermophilus strain with improved anti-inflammatory properties due to the incorporation of the therapeutic cDNA delivery plasmid pValac::il-10. To achieve this purpose, cells of S. thermophilus CRL807, previously selected as being an important anti-inflammatory strain, were electroporated with pValac::il-10 plasmid. In order to confirm the functionality of the developed strain, it was co-cultured with human epithelial cells Caco-2 and the production of IL-10 was evaluated by ELISA. Bacterial suspensions of S. thermophilus CRL807 containing pValac::il-10 plasmid or of the wild-type (WT) strain were administered in vivo using a murine model of intestinal inflammation. The animals treated with S. thermophilus CRL807 pValac::il-10 showed a lower body weight loss, microbial translocation to liver and damage scores in their intestines at macroscopical and microscopic levels. Furthermore, a significant increase was observed in the concentration of IL-10 in the intestinal contents of these mice compared to the rest of the experimental groups, accompanied by decreased levels of pro-inflammatory cytokines. The insertion of the therapeutic pValac::il-10 plasmid increased the intrinsic anti-inflammatory activity (synergetic effect) of S. thermophilus CRL807 which could be included in novel treatment protocols for inflammatory bowel diseases.

Pan-genome analysis of human gastric pathogen H. pylori: comparative genomics and pathogenomics approaches to identify regions associated with pathogenicity and prediction of potential core therapeutic targets.

Helicobacter pylori is a human gastric pathogen implicated as the major cause of peptic ulcer and second leading cause of gastric cancer (~70%) around the world. Conversely, an increased resistance to antibiotics and hindrances in the development of vaccines against H. pylori are observed. Pan-genome analyses of the global representative H. pylori isolates consisting of 39 complete genomes are presented in this paper. Phylogenetic analyses have revealed close relationships among geographically diverse strains of H. pylori. The conservation among these genomes was further analyzed by pan-genome approach; the predicted conserved gene families (1,193) constitute ~77% of the average H. pylori genome and 45% of the global gene repertoire of the species. Reverse vaccinology strategies have been adopted to identify and narrow down the potential core-immunogenic candidates. Total of 28 nonhost homolog proteins were characterized as universal therapeutic targets against H. pylori based on their functional annotation and protein-protein interaction. Finally, pathogenomics and genome plasticity analysis revealed 3 highly conserved and 2 highly variable putative pathogenicity islands in all of the H. pylori genomes been analyzed.

Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Chemical and thermal influence of the [4Fe-4S]2+ cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis.

The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe-4S]2+ cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe-4S]2+ cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe-4S]2+ cluster. UV-vis and fluorescence spectroscopy results demonstrate that between pH3.0 and 4.0 the integrity of the [4Fe-4S]2+ cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45°C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.