Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays.

Lactococcus lactis subsp. lactis NCDO 2118 was recently reported to alleviate colitis symptoms via its anti-inflammatory and immunomodulatory activities, which are exerted by exported proteins that are not produced by L. lactis subsp. lactis IL1403. Here, we used in vitro and in silico approaches to characterize the genomic structure, the safety aspects, and the immunomodulatory activity of this strain. Through comparative genomics, we identified genomic islands, phage regions, bile salt and acid stress resistance genes, bacteriocins, adhesion-related and antibiotic resistance genes, and genes encoding proteins that are putatively secreted, expressed in vitro and absent from IL1403. The high degree of similarity between all Lactococcus suggests that the Symbiotic Islands commonly shared by both NCDO 2118 and KF147 may be responsible for their close relationship and their adaptation to plants. The predicted bacteriocins may play an important role against the invasion of competing strains. The genes related to the acid and bile salt stresses may play important roles in gastrointestinal tract survival, whereas the adhesion proteins are important for persistence in the gut, culminating in the competitive exclusion of other bacteria. Finally, the five secreted and expressed proteins may be important targets for studies of new anti-inflammatory and immunomodulatory proteins. Altogether, the analyses performed here highlight the potential use of this strain as a target for the future development of probiotic foods.

Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002.

BACKGROUND: Studies have detected mis-assemblies in genomes of the species Corynebacterium pseudotuberculosis. These new discover have been possible due to the evolution of the Next-Generation Sequencing platforms, which have provided sequencing with accuracy and reduced costs. In addition, the improving of techniques for construction of high accuracy genomic maps, for example, Whole-genome mapping (WGM) (OpGen Inc), have allow high-resolution assembly that can detect large rearrangements. RESULTS: In this work, we present the resequencing of Corynebacterium pseudotuberculosis strain 1002 (Cp1002). Cp1002 was the first strain of this species sequenced in Brazil, and its genome has been used as model for several studies in silico of caseous lymphadenitis disease. The sequencing was performed using the platform Ion PGM and fragment library (200 bp kit). A restriction map was constructed, using the technique of WGM with the enzyme KpnI. After the new assembly process, using WGM as scaffolder, we detected a large inversion with size bigger than one-half of genome. A specific analysis using BLAST and NR database shows that the inversion occurs between two homology RNA ribosomal regions. CONCLUSION: In conclusion, the results showed by WGM could be used to detect mismatches in assemblies, providing genomic maps with high resolution and allow assemblies with more accuracy and completeness. The new assembly of C. pseudotuberculosis was deposited in GenBank under the accession no. CP012837.

Milk fermented with a 15-lipoxygenase-1-producing Lactococcus lactis alleviates symptoms of colitis in a murine model.

Inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the administration of milks fermented by a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a trinitrobenzenesulfonic acid-induced IBD mouse model. The results obtained demonstrated that 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model. The work also confirmed previous studies showing that fermented milk is an effective form of administration of recombinant lactic acid bacteria expressing beneficial molecules.

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.

Protective effects of lactococci strains delivering either IL-10 protein or cDNA in a TNBS-induced chronic colitis model.

BACKGROUND: Oral treatment with Lactococcus lactis strains secreting the anti-inflammatory cytokine interleukin (IL)-10 has previously shown success as a therapy for inflammatory bowel diseases (IBD). GOALS: Our aim was to compare the protective effects of IL-10, delivered by recombinant lactoccoci using 2 novel expression systems, in a murine colitis model mimicking the relapsing nature of IBD. The first system is based on a Stress-Inducible Controlled Expression system for the production and delivery of heterologous proteins at mucosal surfaces and the second allows the delivery to the host cells of an il-10 cDNA cassette, harbored in a eukaryotic DNA expression vector (pValac). STUDY: Colitis was induced in female BALB/c mice by intrarectal injection of 2,4,6-trinitrobenzenesulphonic acid (TNBS). Mice that recovered received one of the bacteria treatments or saline solution orally during 14 days. Colitis was reactivated 25 days after the first TNBS injection with a second TNBS challenge. Three days after colitis reactivation, cytokine profiles and inflammation in colon samples were evaluated. RESULTS: Animals (N=9) receiving L. lactis strains secreting IL-10 using Stress-Inducible Controlled Expression system or delivering pValac:il-10 plasmid showed lower weight loss (P<0.005), lower damage scores (P<0.005), and immune activation in their large intestines compared with inflamed nontreated mice. CONCLUSIONS: Our results confirm the protective effect of IL-10 delivered either as a protein or as a cDNA in a colitis model mimicking the relapsing nature of IBD and provides a step further in the "proof-of-concept" of genetically engineered bacteria as a valid system to deliver therapeutic molecules at mucosal level.

Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain.

Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity.

Tight controlled expression and secretion of Lactobacillus brevis SlpA in Lactococcus lactis.

Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xylose-inducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle.