Dataset Information

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening.

ABSTRACT:

Background

In-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.

Methods

To identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium (enterotoxigenic E. coli) and a Gram-positive bacterium (Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.

Results

A total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening, with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment. Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression, showing a minimum 10-fold increase in IPEC-J2, 3D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants, but not 3D4/31 cells. In addition to pBD3, other porcine HDP genes such as pBD2, PG1-5, and pEP2C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene- and cell type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3D4/31 macrophages (P < 0.05) against both Gram-negative and Gram-positive bacteria.

Conclusions

Our results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

SUBMITTER: Wang J

PROVIDER: S-EPMC7798283 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening.

Wang Jing J Lyu Wentao W Zhang Wei W Chen Yonghong Y Luo Fang F Wang Yamin Y Ji Haifeng H Zhang Guolong G

Journal of animal science and biotechnology 20210111 1

<h4>Background</h4>In-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.<h4>Methods</h4>To identify natural products with an abili ...[more]

PMID: 33431034

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Project description:Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1?kb porcine ?-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1-5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24?h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

Project description:Despite rapid evolution in the area of microbial natural products chemistry, there is currently no open access database containing all microbially produced natural product structures. Lack of availability of these data is preventing the implementation of new technologies in natural products science. Specifically, development of new computational strategies for compound characterization and identification are being hampered by the lack of a comprehensive database of known compounds against which to compare experimental data. The creation of an open access, community-maintained database of microbial natural product structures would enable the development of new technologies in natural products discovery and improve the interoperability of existing natural products data resources. However, these data are spread unevenly throughout the historical scientific literature, including both journal articles and international patents. These documents have no standard format, are often not digitized as machine readable text, and are not publicly available. Further, none of these documents have associated structure files (e.g., MOL, InChI, or SMILES), instead containing images of structures. This makes extraction and formatting of relevant natural products data a formidable challenge. Using a combination of manual curation and automated data mining approaches we have created a database of microbial natural products (The Natural Products Atlas, www.npatlas.org) that includes 24 594 compounds and contains referenced data for structure, compound names, source organisms, isolation references, total syntheses, and instances of structural reassignment. This database is accompanied by an interactive web portal that permits searching by structure, substructure, and physical properties. The Web site also provides mechanisms for visualizing natural products chemical space and dashboards for displaying author and discovery timeline data. These interactive tools offer a powerful knowledge base for natural products discovery with a central interface for structure and property-based searching and presents new viewpoints on structural diversity in natural products. The Natural Products Atlas has been developed under FAIR principles (Findable, Accessible, Interoperable, and Reusable) and is integrated with other emerging natural product databases, including the Minimum Information About a Biosynthetic Gene Cluster (MIBiG) repository, and the Global Natural Products Social Molecular Networking (GNPS) platform. It is designed as a community-supported resource to provide a central repository for known natural product structures from microorganisms and is the first comprehensive, open access resource of this type. It is expected that the Natural Products Atlas will enable the development of new natural products discovery modalities and accelerate the process of structural characterization for complex natural products libraries.

Dataset Information

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening.

Background

Methods

Results

Conclusions

Publications

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening.

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