Transcriptome response of Salmonella enterica subsp. enterica serovar Typhimurium N-15 and Bifidobacterium thermophilum RBL67 in co-culture
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ABSTRACT: Bifidobacterium thermophilum RBL67 (RBL67), a human fecal isolate and promising probiotic candidate, showed antagonistic and protective effects against Salmonella and Listeria in vitro. However, the underlying mechanisms fostering these health-related effects remain unknown. Therefor the transcriptome response of RBL67 and Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) in co-culture compared to the response in their respective mono-cultures. RNA was extracted from culture samples taken after 4 (N-15) or 5 h (RBL67) and RNAseq was performed on an Illumina HiSeq 2000 sequencer. Three biological replciates were performed resulting in 12 data sets: 3 RBL67 mono culture, 3 N15 mono-culture, 3 RBL67 co-culture, 3 N15 co-culture. Our study provided first insights into probiotic-pathogen interaction on transcriptional level and suggests a mechanism for how probiotic organisms can protect the host from infections. RNA was extracted from culture samples taken after 4 (N-15) or 5 h (RBL67) and RNAseq was performed on an Illumina HiSeq 2000 sequencer. Three biological replciates were performed resulting in 12 data sets: 3 RBL67 mono culture, 3 N15 mono-culture, 3 RBL67 co-culture, 3 N15 co-culture.
Project description:In present study, it was found that the co-culture of Wickerhamomyces anomalus Y-5 and L. plantarum RX-8 could enhance bacteriocin production. To analyze the interaction between W. anomalus Y-5 and L. plantarum RX-8, a quantitative proteomic approach was used to analyze and compare the proteome in L. plantarum RX-8 and W. anomalus Y-5 under mono-culture and co-culture. In total, 339 differently expressed proteins (DEPs) were screened in comparison of L. plantarum RX-8 under mono-culture and co-culture, 645 proteins of W. anomalus Y-5 changed in mono-culture and co-culture. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that DEPs participated in various metabolic pathways such as PTS system, glycolysis, galactose metabolism, glutamate, aspartate, arginine and cysteine metabolism etc. These pathways were related to inducing mechanism on improving bacteriocin production by co-culture. Quantitative proteomic analysis-based strategies can therefore provide further evidence for new regulated targets to improve the production of bacteriocins.
Project description:This study was undertaken with two major goals in mind: i) To investigate the potential for interspecies hydrogen transfer between a hemicellulytic rumen bacterium (B. proteoclasticus) and a methanogenic achaea (M. ruminantium) Microscopic examination had previously shown B. proteoclasticus in co-culture with M. ruminantium rapidly formed cell to cell co-aggregates. ii) To examine the expression of genes involved in methanogenesis under more rumen-like conditions. Cells of M.ruminantium and B. proteoclasticus were grown in triplicate as mono- or co-cultures until a mid-exponential growth phase was reached. RNAs were extracted and purified. The relative quantities of RNAs contributed by each organism to the co-culture samples were determined by rt qPCR using genes previously shown to be constitutively expressed. The mono-culture RNAs were then combined in the determined proportions to normalise mRNA abundance with their co-culture replicates. cDNAs were synthesized and labelled and hybridised to the arrays. 3 biological replicates were used and the cDNA from each was divided in two to allow each to undergo a dyeswap (total of six arrays) giving technical replicates. Each gene was probed in triplicate adding further technical replication. Blanks and negative controls were also included. The log ratios for each probes replicates (biological and technical) were averaged prior to further analysis.
Project description:The aim of the present study was to analyze four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). The mBCECs and pericytes were isolated from brains of adult C57BL/6 mice, and glial cells (mainly astrocytes and microglia) were prepared from cerebral cortices of newborn C57BL/6 mice. The mBCECs were grown as mono-culture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells.
Project description:Porphyromonas gingivalis and Treponema denticola are periodontalpathogens that are associated with the severity and progression of periodontal diseases. This study investigates the gene expression of Porphyromonas gingivalis during co-culture with Treponema denticola
Project description:Porphyromonas gingivalis and Treponema denticola are periodontalpathogens that are associated with the severity and progression of periodontal diseases. this study investigates the gene expression of Treponema denticola during co-culture with Porphyromonas gingivalis.
Project description:Transcriptional profiling of K. vulgare cells co-cultured with Bacillus megaterium compared to K. vulgare mono-cultured cells. Differentially expressed genes in co-cultured and mono-cultured K. vulgare cells were analyzed. The aim was to investigate the mechanisms of B. megaterium stimulating K. vulgare propagation on global gene expression. Two-condition experiment: co-cultured K. vulgare and B. megaterium cells vs. mono-cultured K. vulgare cells. Biological replicates: 3 co-cultured replicates, 3 mono-cultured replicates.
Project description:The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Using iTRAQ-based quantitative assessments, we found that the primary proteins involved in carbohydrate and fatty acid metabolism, adipogenesis and the electron transport chain were highly expressed in 3D cell culture system when compared to those of 2D-cultured cells. Furthermore, it was also shown that the expression levels of proteins associated with metabolic pathways, carbon metabolism and glycolysis/gluconeogenesis were up-regulated, whereas proteins implicated in both DNA replication and the cell cycle were expressed at lower levels compared to those of the 2D mono-cultured cells. Based on these results, the 3D adipocyte model can help elucidate the mechanisms underpinning metabolic syndromes and aid the development of new medical treatments for metabolic disorders.
Project description:The effect of the presence of HMEC-1 cells on HK-2 cell gene expression was investigated. Cells were cultured in mono or co-culture on opposite sides of aluminium oxide filters, 0.2 ?µM pore size. These conditions provide a close proximity but not direct contact. RNA from HK-2 cells was assayed with Affymetrix HGU133 Plus 2 arrays. 3 Biological samples were used per group.
Project description:Expression profiles of 917 pathway repoter genes were determined by AmpliSeq-RNA in primary human hepatocytes treated with Diclofenac and a test compound 3 hours after treatment. Vehicle control, diclofenac, and three doses of the test compound (small-molecule neurotransmitter receptor antagonist) were applied to three primary cell lines, with three biological replicates in each group. In some treatment groups read-outs were only available for two samples. All together 41 samples were profiled.
Project description:Bone marrow derived stromal cells (BMSCs) are a multipotent population that supports angiogenesis, wound healing, immunomodulation and plays an active role in the hematopoietic niche. On the other hand, they are also involved in the nurturing of bone marrow tumors and metastasis, showing a pro-tumorigenic behavior. BMSCs secrete a wide range of cytokines, growth factors and matrix proteins that are likely responsible for many of these effects. However, it is not clear whether this pro-tumorigenic behavior of BMSCs is induced by the tumor cells, neither in what extent the tumor cells affect the type and quantity of factors produced by BMSCs. To determine how tumor cells that arise from bone marrow affect the BMSCs, we selected three myeloid leukemia cell lines (TF-1, TF-1alpha and K562) and co-cultured them with BMSCs from healthy donors. We found that, under co-culture condition, the gene expression profiling of BMSCs revealed up-regulation of many pro-inflammatory signaling related genes, mainly IL-17 signaling-related genes. Moreover, IL-17 signaling-related cytokines CCL2 and IL8, were increased in co-culture supernatants. We conclude that BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profile. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable to leukemia stem cells. BMSCs from healthy donors were transwell co-cultured with three different myeloid leukemia cell lines: TF-1 (n=3), TF-1alpha (n=3) and K562 (n=3). A 1-um Transwell system (BDBiosciences, San Jose, CA USA) was used to maintain the cultured BMSC and leukemia cell populations separate from each other. As a control BMSCs were also transwell co-cultured under the same conditions with CD34+ cells (n=9) isolated from G-CSF-mobilized peripheral blood stem cells from healthy donors. An alternative co-culture method was used to analyze BMSCs and leukemia cells in direct contact: TF-1 (n=3), TF-alpha (n=3) and K562 (n=3). The two populations were cultured together in the same well without any membrane separation. BMSCs (n=18), TF-1 (n=3), TF-1alpha (n=3), K562 (n=3) and CD34+ (n=9) cells cultured alone (mono-cultures) were used as controls. Cells from both mono- and co-culture conditions were harvested at 4h, 10h, and 24h.