ABSTRACT: Genome analysis of Lactobacillus plantarum and Lactobacillus brevis isolated from traditionally fermented Ethiopian kocho and their probiotic properties
Project description:Background: Lactobacillus plantarum is found in a variety of fermented foods and as such, consumed for centuries. Some strains are natural inhabitants of the human gastro-intestinal tract and like other Lactobacillus species, L. plantarum has been extensively studied for its immunomodulatory properties and its putative health-promoting effects (probiotic). Being the first line of host defense intestinal epithelial cells (IEC) are key players in the recognition and initiation of responses to gut microorganisms. Results: Using high-density oligonucleotide microarrays we examined the gene expression profiles of differentiated Caco-2 cells exposed to various doses of L. plantarum. In addition, the effects were correlated to monolayer permeability studies and measurement of lactic acid production. A transcriptional dose-dependent IEC response to L. plantarum was found. Incubation of Caco-2 with a low bacterial dose induced a specific response, not due to cytotoxicity or production of lactic acid, including modulation of cell cycle and cell signaling functions. Exposure of Caco-2 cells to larger amounts of bacteria, accompanied by the production of lactic acid and glucose depletion, provoked increased permeability and supposed non-specific defense responses. Conclusions: These results suggest that IEC are able to sense and react to the presence of gut bacteria. This study provides the first description of global transcriptional response of human IEC to a commensal lactic acid bacterium, and it shows the importance of choosing physiological bacterial doses to prevent the observation of non-specific host reactions. Caco-2 cells were exposed for 10h to Lactobacillus. Fourteen samples are analyzed: 4 control Caco-2, 4 Caco-2 exposed to a low dose (10) of Lactobacillus, 4 Caco-2 exposed to a medium dose (100) of Lactobacillus, 2 Caco-2 exposed to a high dose (1000) of Lactobacillus. All 14 RNA samples are labeled with Cy5 and hybridized to a common reference (undifferentiated Caco-2, untreated) RNA labeled with Cy3
Project description:In order to understand LBG derived galacto-manno-oligosaccharides utilization by a probiotic bacterium, Lactobacillus plantarum WCFS1, we have grown Lactobacillus plantarum WCFS1 (in duplicates) till mid log phase (OD600nm ~0.5, 10 h) in carbon free MRS (de Man, Rogosa Sharpe ) media containing either galacto-manno-oligosaccharides, mannose, glucose or galactose (1% w/v) as the sole carbon source.
Project description:Background: Lactobacillus plantarum is found in a variety of fermented foods and as such, consumed for centuries. Some strains are natural inhabitants of the human gastro-intestinal tract and like other Lactobacillus species, L. plantarum has been extensively studied for its immunomodulatory properties and its putative health-promoting effects (probiotic). Being the first line of host defense intestinal epithelial cells (IEC) are key players in the recognition and initiation of responses to gut microorganisms. Results: Using high-density oligonucleotide microarrays we examined the gene expression profiles of differentiated Caco-2 cells exposed to various doses of L. plantarum. In addition, the effects were correlated to monolayer permeability studies and measurement of lactic acid production. A transcriptional dose-dependent IEC response to L. plantarum was found. Incubation of Caco-2 with a low bacterial dose induced a specific response, not due to cytotoxicity or production of lactic acid, including modulation of cell cycle and cell signaling functions. Exposure of Caco-2 cells to larger amounts of bacteria, accompanied by the production of lactic acid and glucose depletion, provoked increased permeability and supposed non-specific defense responses. Conclusions: These results suggest that IEC are able to sense and react to the presence of gut bacteria. This study provides the first description of global transcriptional response of human IEC to a commensal lactic acid bacterium, and it shows the importance of choosing physiological bacterial doses to prevent the observation of non-specific host reactions. Keywords: host-microbe interaction; dose response; transcriptional analysis
Project description:This project investigated the role of lipoproteins in immune signaling by the model probiotic Lactobacillus plantarum, by mutagenesis of the prolipoprotein diacylglyceryltransferase encoding lgt gene involved in lipoprotein acylation.
Project description:Lactobacillus plantarum WCFS1 was differentially fermented in 29 different fermentations according to a factorial, combinatorial scheme that included variations in growth temperature (28 or 37C), NaCl concentration (0 or 0.3M), pH (5.2, 5.8, 6.4), as well as oxygen (N2 or air) and amino acid availability (1.1 or 2x concentration).
Project description:Housekeeping sigma factors in the Sigma70 family, as components of the RNA polymerase holoenzyme, are responsible for regulating transcription of genes related to vegetative growth. While these factors are well understood in model organisms such as Escherchia coli and Bacillus subtilis, little experimental work has focused on the sigma factors in members of the Lactobacillus genus such as Lactobacillus brevis and Lactobacillus plantarum. This study evaluates the ability of putative Sigma70 proteins from L. brevis (Sigma70-Lb) and L. plantarum (Sigma70-Lp) to complement a temperature sensitive mutation in E. coli 285c Sigma70. After finding that the heterologous sigma factors were capable of restoring the viability of E. coli 285c at 42 C through growth kinetics studies, the transcriptional responses of 285c to an extended heat shock in the presence of Sigma70-Lb and Sigma70-Lp were found to be similar to previous studies. These results indicate the Sigma70-Lb and Sigma70-Lp are capable of initiating transcription in a complex with the E. coli 285c RNA polymerase to a sufficient degree to restore viability at elevated temperatures without triggering unusual modifications to the native transcriptional program. These heterologous sigma factors may therefore be useful to improve biochemical knowledge of the sigma factor family or for use in transcriptional engineering.
Project description:Housekeeping sigma factors in the Sigma70 family, as components of the RNA polymerase holoenzyme, are responsible for regulating transcription of genes related to vegetative growth. While these factors are well understood in model organisms such as Escherchia coli and Bacillus subtilis, little experimental work has focused on the sigma factors in members of the Lactobacillus genus such as Lactobacillus brevis and Lactobacillus plantarum. This study evaluates the ability of putative Sigma70 proteins from L. brevis (Sigma70-Lb) and L. plantarum (Sigma70-Lp) to complement a temperature sensitive mutation in E. coli 285c Sigma70. After finding that the heterologous sigma factors were capable of restoring the viability of E. coli 285c at 42 C through growth kinetics studies, the transcriptional responses of 285c to an extended heat shock in the presence of Sigma70-Lb and Sigma70-Lp were found to be similar to previous studies. These results indicate the Sigma70-Lb and Sigma70-Lp are capable of initiating transcription in a complex with the E. coli 285c RNA polymerase to a sufficient degree to restore viability at elevated temperatures without triggering unusual modifications to the native transcriptional program. These heterologous sigma factors may therefore be useful to improve biochemical knowledge of the sigma factor family or for use in transcriptional engineering. 3 biological replicates per sigma factor
Project description:Background: In the last decade, much attention has been drawn to probiotic bacteria in the context of inflammatory bowel disease (IBD), since the potential of certain strains to attenuate inflammation was demonstrated in several animal experiments and clinical studies. Data in humans elucidating the molecular mechanism of probiotic action are still scarce. To this end, we used an organ culture system of human colon mucosa and investigated the gene expression profiles after treatment with different probiotic bacteria in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)) stimulated samples using whole genome microarrays. Moreover, we analyzed changes occurring in the intestinal explants cultured for 8 hours when compared to fresh, directly frozen mucosa, in order to infer the suitability of the system to study an inflammatory stimulus and likely antiinflammatory responses. Results: Culturing intestinal colon fragments during 8 hours elicited differential gene expression in 283 genes, 229 upregulated and 54 downregulated. Upregulated genes were predominantly related to apoptosis, whereas downregulated genes encoded mitochondrial proteins. No specific enrichment of genes related to inflammation or immune response could be detected, confirming the suitability of the system to further study the inmunomodulatory/anti-inflammatory properties of Lactobacillus casei BL23 (BL23), L.plantarum 299v (LP299v) and L.plantarum 299v (A-) (LP299v (A-)), a mutant strain with reduced adhesive properties to enterocytes. Intestinal explants were stimulated with PMA/IO for 3 hours and subsequently incubated with probiotic bacteria for 4 h. ANOVA analysis (p ? 0,01) revealed 205 differentially expressed genes between Control, PMA/IO (Inflamed), and the 3 bacterial treatments. Most importantly, a number of PMA/IO induced genes related to immune response and immune system process such as IL-2, IFN-?, IL17A and pro-inflammatory cytokines CXCL9 and CXCL11 were downregulated by BL23, LP299v and LP299v (A-). The behaviour of the three Lactobacillus strains was quite similar, although their presence induced differential expression of a small number of genes in a strain dependent manner. Conclusion: The human colon organ culture was found to be a suitable model for the study of inflammatory/anti-inflammatory stimuli, and therefore it constitutes a valuable tool to determine the inmunomodulatory effect of probiotic bacteria. The global transcriptional profile evoked by strains BL23, LP299v and LP299v (A-) in artificially inflamed tissue indicated a clear homeostasis restoring effect, including a decrease of the signals produced by activated T cells. Macroscopically healthy colonic intestinal tissue was obtained at surgery from 3 patients. Intestinal explants were treated with PMA and ionomycin for 3 h to induce pro-inflammatory conditions. Then, culture medium was changed and replaced with either medium or medium containing either Lactobacillus casei BL23, Lactobacillus plantarum 299v, or a nonadherent mutant of L. plantarum 299v (A-) and incubated for further 4 hours. In parallel, control intestinal explants were cultured without any treatment of PMA/ionomycin or probiotic bacteria and compared to directly frozen tissue in order to evaluate changes in gene expression which are due solely to the culture conditions.