Unknown,Transcriptomics,Genomics,Proteomics

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Differential Immunomodulatory Impact of Bacillus subtilis Spores and Vegetative Cells on a Human Colonic Epithelial Cell-Line Model

ABSTRACT: Bacillus subtilis strain R0179 is found in a number of commercially-available probiotic products. The mechanism(s) of action of B. subtilis in the host are poorly understood, but may involve the immune system response to switching between spore and vegetative forms. In order to help elucidate this mechanism, we challenged the immune response of a human colonic epithelial HT-29 cell model for 3 hours with the two forms of B. subtilis. The cellular response was evaluated using a custom-designed two-color expression microarray targeting 1354 genes of the human immune system. The data obtained in this study indicates that the vegetative cell form of the strain moderately induced TH1 pro-inflammatory response through IL-17C and TNF signaling pathway while down-regulating anti-inflammatory response genes IL-10 and TGFÎ²-2. The spore form had an opposite effect and acted primarily by down-regulating the Mitogen-Activated Protein Kinase pathway. The overall design consisted of 2 samples of HT-29 cells treated with Bacillus subtilis R0179 spores or vegetative states versus unchallenged HT-29 cells. A minimum of four dye-swap hybridizations (4 biological replicates) were performed for each of the 2 samples analyzed. Unchallenged HT-29 cells were controls and challenged HT-29 cells with Bacillus subtilis R0179 spores or vegetative were treated samples.

ORGANISM(S): Homo sapiens

SUBMITTER: Thomas Tompkins

PROVIDER: E-GEOD-60412 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Description Bacillus subtilis forms highly resistant, metabolically inactive spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants through germination and outgrowth, and then develop into vegetative cells. However, the mechanism of the activation of the molecular machinery that triggers spore germination and outgrowth is unclear. To gain further insight into spore germination and outgrowth, the transcriptome and proteome changes during Bacillus subtilis spore conversion to vegetative cells were analyzed. The transcriptome analysis also allowed us to trace the different functional groups of genes expressed during this conversion. For each time-point sampled, the change in the spore proteome was quantitatively monitored relative to the reference proteome of 15N metabolically labelled vegetative cells. Of the quantified proteins, 60 percent are common to vegetative cells and spores, indicating that spores have a minimal set of proteins sufficient for the resumption of metabolism upon completion of germination. The shared proteins thus represent the most basic survival kit for spore-based life. Until the phase transition, defined as the completion of germination, we observed no significant change in the proteome or the transcriptome. Our analysis identified 34 abundant mRNA transcripts in the dormant spores, at least 31 of which were rapidly degraded after the phase transition. We observed 3152 differentially expressed genes, and demonstrated with our mass spectrometry analyses the differential expression of 323 proteins. Our data show that 173 proteins from dormant spores, both proteins unique to spores and proteins shared with vegetative cells, are lost after completion of germination. Further analysis is required to functionally interpret the observed protein loss. The observed diverse timing of the synthesis of different protein sets reveals a putative core-strategy of the revival of life starting from the B. subtilis spore.

Project description:A commercially available product containing three probiotic bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033, and Bifidobacterium bifidum R0071) was previously shown in animal trials to modulate both TH1 and TH2 immune responses. Clinical studies on this combination of bacteria have also shown positive health effects against seasonal winter diseases and rotavirus infection. The goal of this study was to use a well-established in vitro intestinal epithelial (HT-29) cell model that has been shown to constitutively express double-stranded RNA (dsRNA) sensors (Toll-like receptor 3[TLR3], retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and dsRNA-activated protein kinase). By using the HT-29 cell model, we wanted to evaluate whether or not this combination of three bacteria had the capacity to immune modulate the host cell response to a dsRNA ligand, poly(IM-bM-^@M-"C). Using a custom-designed, two-color expression microarray targeting genes of the human immune system, we investigated the response of HT-29 cells challenged with poly(IM-bM-^@M-"C) both in the presence and in the absence of the three probiotic bacteria. We observed that the combination of the three bacteria had a major impact on attenuating the expression of genes connected to proinflammatory TH1 and antiviral innate immune responses compared to that obtained by the poly(IM-bM-^@M-"C)-only challenge. Major pathways through which the multistrain combination may be eliciting its immune-modulatory effect include the TLR3-TRIF, mitogen-activated protein kinase, and NF-KB signaling pathways.Such a model may be useful for selecting potential biomarkers for the design of future clinical trials The overall design consisted of 3 samples of HT-29 cells treated with poly (I:C)-only, probiotic combination (PC) and poly (I:C) + PC versus unchallenged HT-29 cells. A minimum of four dye-swap hybridizations (4 biological replicates) were performed for each of the 3 samples analyzed. Unchallenged HT-29 cells were controls and challenged HT-29 cells with poly (I:C)-only, PC-only and poly (I:C) + PC were treated samples.

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Differential Immunomodulatory Impact of Bacillus subtilis Spores and Vegetative Cells on a Human Colonic Epithelial Cell-Line Model

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