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Rainbow Trout (Oncorhynchus Mykiss) Intestinal Epithelial Cells as a Model for Studying Gut Immune Function and Effects of Functional Feed Ingredients

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The objective of this study was to evaluate the suitability of the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model for studies of gut immune function and effects of functional … feed ingredients. Effects of lipopolysaccharide (LPS) and three functional feed ingredients [nucleotides, mannanoligosaccharides (MOS), and beta-glucans] were evaluated in RTgutGC cells grown on conventional culture plates and transwell membranes. Permeation of fluorescently-labeled albumin, transepithelial electrical resistance (TEER), and tight junction protein expression confirmed the barrier function of the cells. Brush border membrane enzyme activities [leucine aminopeptidase (LAP) and maltase] were detected in the RTgutGC cells but activity levels were not modulated by any of the exposures. Immune related genes were expressed at comparable relative basal levels as these in rainbow trout distal intestine. LPS produced markedly elevated gene expression levels of the pro-inflammatory cytokines il1b, il6, il8, and tnfa but had no effect on ROS production. Immunostaining demonstrated increased F-actin contents after LPS exposure. Among the functional feed ingredients, MOS seemed to be the most potent modulator of RTgutGC immune and barrier function. MOS significantly increased albumin permeation and il1b, il6, il8, tnfa, and tgfb expression, but suppressed ROS production, cell proliferation and myd88 expression. Induced levels of il1b and il8 were also observed after treatment with nucleotides and beta-glucans. For barrier function related genes, all treatments up-regulated the expression of cldn3 and suppressed cdh1 levels. Beta-glucans increased TEER levels and F-actin content. Collectively, the present study has provided new information on how functional ingredients commonly applied in aquafeeds can affect intestinal epithelial function in fish. Our findings suggest that RTgutGC cells possess characteristic features of functional intestinal epithelial cells indicating a potential for use as an efficient in vitro model to evaluate effects of bioactive feed ingredients on gut immune and barrier functions and their underlying cellular mechanisms.

Authors: Jie Wang, Peng Lei, Amr Ahmed Abdelrahim Gamil, Leidy Lagos, Yang Yue, Kristin Schirmer, Liv Torunn Mydland, Margareth Overland, Åshild Krogdahl, Trond M. Kortner

Date Published: 6th Feb 2019

Publication Type: Journal

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

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Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a … hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Authors: Z. Xu, Y. Wang, K. F. Chater, H. Y. Ou, H. H. Xu, Z. Deng, M. Tao

Date Published: 8th Jan 2017

Publication Type: Not specified

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