In vivo expression of the Lactobacillus brevis S-layer gene.
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ABSTRACT:
SUBMITTER: Kahala M
PROVIDER: S-EPMC178692 | biostudies-other | 1997 Jan
REPOSITORIES: biostudies-other
Publications
In vivo expression of the Lactobacillus brevis S-layer gene.
Kahala M M Savijoki K K Palva A A
Journal of bacteriology 19970101 1
Lactobacillus brevis possesses a surface layer protein (SlpA) with tightly regulated synthesis. The slpA gene is expressed by two adjacent promoters, P1 and P2. The level of P2-derived transcripts was approximately 10 times higher than that of P1-derived transcripts throughout the entire growth of L. brevis. The half-lives of slpA transcripts were shown to be exceptionally long (14 min). ...[more]
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Project description:So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.
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Project description:The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates poses significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Currently, these inhibitory compounds must be removed through additional downstream processing to create feedstock suitable for most industrially important microbial strains. This study explores the high ferulic acid tolerance in Lactobacillus brevis (L. brevis), a lactic acid bacteria often found in fermentation processes, by global transcriptional response analysis. The transcriptional profile of L. brevis under ferulic acid stress reveals that the presence of ferulic acid primarily triggers the expression of membrane proteins to counteract ferulic acid induced changes in membrane fluidity and ion leakage, in the midst of a generalized stress response. Several promising routes for understanding phenolic acid tolerance have been identified based upon these findings. These insights may be used to guide further engineering of model industrial organisms to better tolerate phenolic compounds in processed biomass.
2011-12-31 | GSE22826 | GEO
Project description:C-type lectin receptors (CLRs) are pattern recognition receptors that are crucial in the innate immune response. The gastrointestinal tract contributes significantly to the maintenance of immune homeostasis; it is the shelter for billions of microorganisms including many genera of Lactobacillus sp. Previously, it was shown that host-CLR interactions with gut microbiota play a crucial role in this context. The Macrophage-inducible C-type lectin (Mincle) is a Syk-coupled CLR that contributes to sensing of mucosa-associated commensals. In this study, we identified Mincle as a receptor for the Surface (S)-layer of the probiotic bacteria Lactobacillus brevis modulating GM-CSF bone marrow-derived cells (BMDCs) functions. We found that the S-layer/Mincle interaction led to a balanced cytokine response in BMDCs by triggering the release of both pro- and anti-inflammatory cytokines. In contrast, BMDCs derived from Mincle-/-, CARD9-/- or conditional Syk-/- mice failed to maintain this balance, thus leading to an increased production of the pro-inflammatory cytokines TNF and IL-6, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-β were markedly decreased. Importantly, this was accompanied by an altered CD4+ T cell priming capacity of Mincle-/- BMDCs resulting in an increased CD4+ T cell IFN-γ production upon stimulation with L. brevis S-layer. Our results contribute to the understanding of how commensal bacteria regulate antigen-presenting cell (APC) functions and highlight the importance of the Mincle/Syk/Card9 axis in APCs as a key factor in host-microbiota interactions.
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Project description:Lactic acid bacteria confer a variety of health benefits. Here we investigate the mechanisms by which Lactobacillus brevis KB290 enhances cell-mediated cytotoxic activity. We fed a diet containing KB290 (3 × 10^9 colony-forming units/g) , or potato starch, to 9-week-old female BALB/c mice for 1, 4, 7, or 14 days and examined the cytotoxic activity of splenocytes was measured. RNA was extracted from the spleen and analyzed for gene expression by DNA microarray. KB290 enhanced the cell-mediated cytotoxic activity of splenocytes. The Gene Ontology (GO) terms related to immune process were significantly enriched in up-regulated gene set for 7 days and the GO terms related to cellular process were enriched in down-regulated gene set on days 4 and 7. Among the GO terms related to immune process, positive regulation of T cell-mediated cytotoxicity was existed. Almost all of the genes included in the term encoded MHC class I molecules, which are involved in the presentation of antigen to CD8+ cytotoxic T cells. On the other hand, the over-represented Kyoto Encyclopedia of Genes and Genomes pathways among the up-regulated genes were those for natural killer cell-mediated cytotoxicity and antigen processing and presentation on day 1. Although the up-regulated genes involved in antigen processing and presentation for stimulation of CD8+ cytotoxic T cells were not observed on day 14, some genes involved in T cell and natural killer cell activation remained up-regulated until day 14. The sequential gene expression profiling reflected the changes in cytotoxic activity during KB290 feeding. These results suggest that the enhanced cytotoxic activity could have been caused both by activation of natural killer cells and by CD8+ cytotoxic T cells stimulated via MHC class I presentation.
2013-08-05 | GSE41127 | GEO
Project description:Lactobacillus brevis has been widely used in industry for fermentation purposes. However, it is also associated with the spoilage of foods and beverages, in particular, beer. There is an increasing demand for natural food preservation methods, and in this context, bacteriophages possess the potential to control such spoilage bacteria. Just a few studies on phages infecting Lactobacillus brevis have been performed to date and in the present study, we report the isolation and characterization of five virulent phages capable of infecting Lb. brevis strains. The analysis reveals a high diversity among the isolates, with members belonging to both, the Myoviridae and Siphoviridae families. One isolate, designated phage 3-521, possesses a genome of 140.8 kb, thus representing the largest Lb. brevis phage genome sequenced to date. While the isolated phages do not propagate on Lb. brevis beer-spoiling strains, phages showed activity against these strains, impairing the growth of some Lb. brevis strains. The results highlight the potential of bacteriophage-based treatments as an effective approach to prevent bacterial spoilage of beer.
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| S-EPMC6805780 | biostudies-literature