Dataset Information

Gene silencing of porcine MUC13 and ITGB5: candidate genes towards Escherichia coli F4ac adhesion.

ABSTRACT:

Background

Integrin beta-5 (ITGB5) and mucin 13 (MUC13) genes are highly expressed on the apical surface of intestinal epithelia and are thought to be candidate genes for controlling the expression of the receptor for enterotoxigenic Escherichia coli (ETEC) F4ac. Human MUC13 protein has an expected role in protecting intestinal mucosal surfaces and porcine ITGB5 is a newly identified potential receptor for ETEC F4ac.

Methodology/principal findings

To test the hypothesis that ITGB5 and MUC13 both play key roles in protection of the intestinal mucosa against pathogenic bacterium, porcine intestinal epithelial cells (IPEC-J2) were transfected with ITGB5-targeting, MUC13-targeting or negative control small interfering RNA (siRNA), respectively. Firstly, we measured mRNA expression levels of mucin genes (MUC4, MUC20), pro-inflammatory genes (IL8, IL1A, IL6, CXCL2), anti-inflammatory mediator SLPI, and PLAU after RNAi treatments with and without ETEC infection. Secondly, we compared the adhesions of ETEC to the pre- and post-knockdown IPEC-J2 cells of ITGB5 and MUC13, respectively. We found that ITGB5 and MUC13 knockdown both had small but significant effects in attenuating the inflammation induced by ETEC infection, and both increased bacterial adhesion in response to F4ac ETEC exposure.

Conclusions/significance

Our current study first reported that ITGB5 and MUC13 are important adhesion molecules of mucosal epithelial signaling in response to Escherichia coli in pigs. These data suggest that both ITGB5 and MUC13 play key roles in defending the attachment and adhesion of ETEC to porcine jejunal cells and in maintaining epithelial barrier and immunity function.

SUBMITTER: Zhou C

PROVIDER: S-EPMC3726385 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Gene silencing of porcine MUC13 and ITGB5: candidate genes towards Escherichia coli F4ac adhesion.

Zhou Chuanli C Liu Zhengzhu Z Liu Yang Y Fu Weixuan W Ding Xiangdong X Liu Jianfeng J Yu Ying Y Zhang Qin Q

PloS one 20130729 7

<h4>Background</h4>Integrin beta-5 (ITGB5) and mucin 13 (MUC13) genes are highly expressed on the apical surface of intestinal epithelia and are thought to be candidate genes for controlling the expression of the receptor for enterotoxigenic Escherichia coli (ETEC) F4ac. Human MUC13 protein has an expected role in protecting intestinal mucosal surfaces and porcine ITGB5 is a newly identified potential receptor for ETEC F4ac.<h4>Methodology/principal findings</h4>To test the hypothesis that ITGB5 ...[more]

PMID: 23922972

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Project description:Enterotoxigenic Escherichia coli (ETEC) that expresses F4ac fimbriae is the major pathogenic microorganism responsible for bacterial diarrhea in neonatal piglets. The susceptibility of piglets to ETEC F4ac is determined by a specific receptor on the small intestinal epithelium surface. We performed an iTRAQ-labeled quantitative proteome analysis using a case-control design in which susceptible and resistant full-sib piglets were compared for the protein expression levels. Two thousand two hundred forty-nine proteins were identified, of which 245 were differentially expressed (fold change > 1.5, FDR-adjusted P < 0.05). The differentially expressed proteins fell into four functional classes: (I) cellular adhesion and binding, (II) metabolic process, (III) apoptosis and proliferation, and (IV) immune response. The integrin signaling pathway merited particular interest based on a pathway analysis using statistical overexpression and enrichment tests. Genomic locations of the integrin family genes were determined based on the most recent porcine genome sequence assembly (Sscrofa11.1). Only one gene, ITGB5, which encodes the integrin ?5 subunit that assorts with the ?v subunit to generate integrin ?v?5, was located within the SSC13q41 region between 13:133161078 and 13:139609422, where strong associations of markers with the ETEC F4ac susceptibility were found in our previous GWAS results. To identify whether integrin ?v?5 is the ETEC F4acR, we established an experimental model for bacterial adhesion using IPEC-J2 cells. Then, the ITGB5 gene was knocked out in IPEC-J2 cell lines using CRISPR/Cas9, resulting in a biallelic deletion cell line (ITGB5 -/-). Disruption of ITGB5 significantly reduced ETEC F4ac adhesion to porcine intestinal epithelial cells. In contrast, overexpression of ITGB5 significantly enhanced the adhesion. A GST pull-down assay with purified FaeG and ITGB5 also showed that FaeG binds directly to ITGB5. Together, the results suggested that ITGB5 is a key factor affecting the susceptibility of piglets to ETEC F4ac.

Project description:The development of an early complex gut microbiota may play an important role in the protection against intestinal dysbiosis later in life. The significance of the developed microbiota for gut barrier functionality upon interaction with pathogenic or beneficial bacteria is largely unknown. The transcriptome of differently perfused jejunal loops of 12 caesarian-derived pigs, neonatally associated with microbiota of different complexity, was studied. Piglets received pasteurized sow colostrum at birth (d0), a starter microbiota (Lactobacillus amylovorus (LAM), Clostridium glycolicum, and Parabacteroides) on d1-d3, and a placebo inoculant (simple association, SA) or an inoculant consisting of sow's diluted feces (complex association, CA) on d3-d4. On d 26-37, jejunal loops were perfused for 8 h with either enterotoxigenic Escherichia coli F4 (ETEC), purified F4 fimbriae, LAM or saline control (CTRL). Gene expression of each intestinal loop was analyzed by Affymetrix Porcine Gene 1.1_ST array strips. Gene Set Enrichment Analysis was performed on expression values. Compared to CTRL, 184 and 74; 2 and 139; 2 and 48 gene sets, were up- and down-regulated by ETEC, F4 and LAM, respectively. ETEC up-regulated networks related to inflammatory and immune responses, RNA processing, and mitosis. There was a limited overlap in up-regulated gene sets between ETEC and F4 fimbriae. LAM down-regulated genes related to inflammatory and immune responses, as well as to cellular compound metabolism. In CA pigs, 57 gene sets were up-regulated by CA, while 73 were down-regulated compared to SA. CA up-regulated gene sets related to lymphocyte modulation and to cellular defense in all loop perfusions. In CA pigs, compared to SA pigs, genes for chemokine and cytokine activity and for response to external stimuli were down-regulated in ETEC-perfused loops and up-regulated in CTRL. The results highlight the importance of the nature of neonatal microbial colonization in the response to microbial stimuli later in life.

Project description:Background:Diarrhea represents one of the most frequent major problems during piglets' neonatal and post-weaning periods leading to tremendous economic losses in the swine industry. Enterotoxigenic Escherichia coli (ETEC) F4 is regarded as the most important cause of diarrhea in piglets. However, some pigs are naturally resistant to those diarrheas caused by ETEC-F4, because they have no F4 receptors (F4R) on their small intestine epithelial cells that allow F4 fimbriae attachment. Thus, our study characterized a complete transcriptome of small intestine epithelial cells of Large White piglets using RNA-Seq. The aim of the study was to identify DEGs with regard to differences in the F4R phenotypes and SNP (C/T) genotypes at ITGB5 and important pathways associated with ETEC-F4ac susceptibility in small intestine epithelial cells of Large White piglets and derive molecular markers as a result of loss of F4acR in swine. Methods:A total of eight samples of small intestine epithelial cells obtained from Large White piglets (35 days old) used in this study were selected on the basis of two criteria. One was the adhesion phenotype to ETEC-F4ac fimbriae, and the other was the comparison of ITGB5 SNP (C > T) genotype sequences across all the samples. The samples were then divided into two groups, non-adhesive with CC genotype (n = 4), and adhesive with TT genotype (n = 4). Results:More down-regulated DEGs (p < 0.05, |log2FC| > 2) were detected in the comparison of non-adhesive vs. adhesive small intestine epithelial cells in the present study. Six genes, of which two (CNGA4, SLC25A31) exclusively expressed and four (HCN4, MYLK, KCNMA1, and KCNMB1) DEGs with up-regulation pattern in adhesive (F4R positive) pigs were involved in two pathways associated with diarrhea. The DEGs with up-regulation pattern in non-adhesive (F4R negative) pigs were mostly engaged in multiple immune response-related pathways. Conclusion:The results provide insights on the biology of the phenotypes of F4R positive and negative pigs. One gene (MYLK) located on SSC13 locus for F4acR strongly support that it might have played a role in the adhesion phenotype which was obviously detected by adhesion assay in adhesive (F4R positive) group.

Project description:BackgroundLong non-coding RNAs (lncRNAs) play crucial roles in gene regulation at the transcriptional and post-transcriptional levels. LncRNAs are belonging to a large class of transcripts with ≥200 nt in length which do not code for proteins, have been widely investigated in various physiological and pathological contexts by high-throughput sequencing techniques and bioinformatics analysis. However, little is known about the regulatory mechanisms by which lncRNAs regulate genes that are associated with Enterotoxigenic Escherichia coli F4 fimbriae (ETEC-F4ac) adhesion phenotype in small intestine epithelial cells of Large White piglets. To address this, we used RNA sequencing to profile lncRNAs and mRNAs of small intestine epithelial cells in Large White piglets differing in their ETEC-F4 adhesion phenotypes and ITGB5 genotypes. Eight male piglets were used in this study and were divided into two groups on the basis of their adhesion phenotype and ITGB5 genotypes, a candidate gene for F4ac receptor. Non-adhesive group (n = 4) with CC genotype and adhesive group (n = 4) with TT genotype.ResultsIn total, 78 differentially expressed lncRNAs (DE-lncRNA) and 223 differentially expressed mRNAs (log2 |FC| > 1, P < 0.05) were identified in the comparison of non-adhesive vs. adhesive small intestine epithelial cells. Furthermore, cis- and trans-regulatory target genes of DE-lncRNAs were identified, then interaction networks of lncRNAs and their cis- and trans-target differentially expressed genes (DEGs) were constructed separately. A total of 194 cis-targets were involved in the lncRNAs-cis genes interaction network and 61 trans-targets, were involved in lncRNA-trans gene interaction network that we constructed. We determined that cis-target genes were involved in alcoholism, systemic lupus erythematosus, viral carcinogenesis and malaria. Whereas trans-target DEGs were engaged in three important pathways related to the ETEC-F4 adhesion phenotype namely cGMP-PKG signaling pathway, focal adhesion, and adherens junction. The trans-target DEGs which directly involved in these pathways are KCNMB1 in cGMP-PKG signaling pathway, GRB2 in focal adhesion pathway and ACTN4 in focal adhesion and adherens junction pathways.ConclusionThe findings of the current study provides an insight into biological functions and epigenetic regulatory mechanism of lncRNAs on porcine small intestine epithelial cells adhesion to ETEC-F4-ac and piglets' diarrhea susceptibility/resistance.

Dataset Information

Gene silencing of porcine MUC13 and ITGB5: candidate genes towards Escherichia coli F4ac adhesion.

Background

Methodology/principal findings

Conclusions/significance

Publications

Gene silencing of porcine MUC13 and ITGB5: candidate genes towards Escherichia coli F4ac adhesion.

Similar Datasets

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