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The C-terminal region of Bacteroides fragilis toxin is essential to its biological activity.

ABSTRACT: To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

SUBMITTER: Sears CL

PROVIDER: S-EPMC1594940 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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The C-terminal region of Bacteroides fragilis toxin is essential to its biological activity.

Sears Cynthia L CL Buckwold Simy L SL Shin Jai W JW Franco Augusto A AA

Infection and immunity 20061001 10

To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activit ...[more]

PMID: 16988234

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Project description:Purpose: The goal of this study is to determine how BFT2 alters chromatin accessibility at relatively early time points in colon epithelial cells, and to correlate the changes in chromatin accessibility with changes in gene expression, transcription factor binding sites, and the location of common single nucleotide variants (SNVs) and differentially methylated regions (DMRs) in colorectal cancer. Methods: HT29/C1 cells were plated at low density and allowed to grow for 4 days at 37C and 10% CO2. Afterwards, cells were either left untreated or treated with 100ng/mL BFT2 for 24 or 48 hours. After the specified time point, cells were counted and DNA was collected in order to prepare ATAC-seq libraries. ATAC-seq library was prepared as outlined by Buenrostro et al. (2013). The 4nM pooled ATAC-seq library was sequenced using the Illumina HiSeq. For each sample, three biological replicates were sequenced. After sequencing, the data was analyzed using the pipeline developed by the Kundaje lab, as outlined on ENCODE (Lee et al., 2016). Briefly, reads were trimmed, aligned and filtered using Bowtie2, and peaks were called using MACS2. The three replicate peak files were combined in order to create one consensus file for each treatment condition using an irreproducible discovery rate (IDR) threshold of 0.1. For all analyses, the “optimal set” consensus file was used. Results: Our data show that several genes are differentially expressed after BFT treatment and these changes correlate with changes in chromatin accessibility. This correlation is mediated by an increase in chromatin accessibility at particular transcription factor binding sites. Also, sites of increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC. Conclusions: This study adds critical knowledge to our understanding of host-microbe interactions in the gut. While scientists have begun to analyze the effect of specific bacteria on the epigenome of colon epithelial cells, to date, no other studies have surveyed the effect of a specific bacterial toxin on chromatin accessibility in colon epithelial cells. We conclude that BFT2 alters chromatin accessibility in colon epithelial cells, and that these changes correlate with subsequent changes in gene expression. Also, sites of BFT2-induced increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC.

Project description:Purpose: The goal of this study is to determine how BFT2 alters chromatin accessibility at relatively early time points in colon epithelial cells, and to correlate the changes in chromatin accessibility with changes in gene expression, transcription factor binding sites, and the location of common single nucleotide variants (SNVs) and differentially methylated regions (DMRs) in colorectal cancer. Methods: HT29/C1 cells were plated at low density and allowed to grow for 4 days at 37C and 10% CO2. Afterwards, cells were either left untreated or treated with 100ng/mL BFT2 for 24 or 48 hours. After the specified time point, cells were counted and RNA was collected in order to prepare RNA-seq libraries. RNA was collected using the Qiagen RNeasy mini kit. mRNA was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module. Afterwards, a non-directional RNA-seq library was constructed using the NEBnext Ultra RNA Library Prep kit from Illumina. The 2nM pooled RNA-seq library was sequenced using the Illumina HiSeq. Results: After sequencing, Kallisto was used to perform pseudoalignment of the raw RNA-seq data. Then, Sleuth was used to quantify gene expression and perform differential expression analyses. After treatment with BFT2 for 24 hours, 70 genes were differentially expressed (P-value < 0.01). Of these genes, 41 showed a decrease in gene expression, while 29 showed an increase in gene expression. After BFT2 treatment for 48 hours, we found only 16 differentially expressed genes (P-value < 0.01); 3 showed a decrease in gene expression and 13 showed an increase in gene expression. Conclusions: This study adds critical knowledge to our understanding of host-microbe interactions in the gut. While scientists have begun to analyze the effect of specific bacteria on the epigenome of colon epithelial cells, to date, no other studies have surveyed the effect of a specific bacterial toxin on chromatin accessibility in colon epithelial cells. We conclude that BFT2 alters chromatin accessibility in colon epithelial cells, and that these changes correlate with subsequent changes in gene expression. Also, sites of BFT2-induced increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC.

Project description:Bacteroidales are the most abundant Gram-negative bacteria of the healthy human colonic microbiota, comprising nearly 50% of the colonic bacteria in many individuals. Numerous species and strains of gut Bacteroidales are present simultaneously at high concentrations in this ecosystem. Studies are revealing that gut Bacteroides has numerous antibacterial weapons to antagonize closely related members. In this study, we identify a new diffusible antibacterial toxin produced by Bacteroides fragilis 638R, designated BSAP-4. This is the fifth antibacterial toxin produced by this strain and the second toxin of this strain with a membrane attack complex/perforin domain (MACPF). We identify the target molecule of sensitive cells as a ?-barrel outer membrane protein (OMP) with calycin-like domains. As with other MACPF toxins, the gene encoding the target in sensitive strains is in the same genetic region as bsap-4 in producing strains. A comparison of B. fragilis strains showed there are two sensitive variants of this OMP that are 87% similar to each other and 50% similar to the resistant OMP. Unlike other MACPF toxins, there are numerous B. fragilis strains that harbor the resistant OMP without bsap-4 Several OMP variants from strains that are BSAP-4 resistant under the conditions of our assay confer BSAP-4 sensitivity to Bacteroides thetaiotaomicron when constitutively expressed. Using a reporter assay, we show that the BSAP-4 receptor gene is differentially expressed in sensitive and resistant strains leading to apparent BSAP-4 resistance under the conditions of our assay, despite harboring the BSAP-4 target gene.IMPORTANCE The intestinal microbiota is a diverse microbial ecosystem that provides numerous benefits to humans. The factors that govern its establishment and stability are just beginning to be elucidated. Identification and characterization of antimicrobial toxins produced by its members and their killing range are essential to understanding the role of antagonism in community composition and stability. Here, we identify a fifth antimicrobial toxin produced by a single Bacteroides fragilis strain and identify its target. The finding of such a large number of toxins that antagonize competing members suggests that this feature substantially contributes to the fitness of these bacteria. In addition, these toxins may have applications in genetically engineered gut bacteria to allow engraftment or to antagonize a potentially pathogenic member.

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The C-terminal region of Bacteroides fragilis toxin is essential to its biological activity.

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The C-terminal region of Bacteroides fragilis toxin is essential to its biological activity.

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