title: Bacterial toxin-inducible gene expression of cathelicidin-B1 in the chicken bursal lymphoma-derived cell line DT40: functional characterization of cathelicidin-B1.
abstract: Chicken cathelicidin-B1 (chCATH-B1) is a major host defense peptide of the chicken bursa of Fabricius (BF). To investigate the mechanisms of chCATH-B1 gene expression in the BF, we focused on the DT40 cell line derived from chicken bursal lymphoma as a model for analysis. A cDNA encoding chCATH-B1 precursor was cloned from DT40 cells. The nucleotide sequence of the cDNA was identical with that of the BF chCATH-B1. A broth dilution analysis showed that the synthetic chCATH-B1 exhibited a significant defensive activity against both Escherichia coli and Staphylococcus aureus. A scanning microscopic analysis demonstrated that chCATH-B1 inhibited bacterial growth through membrane destruction with formation of blebs and spheroplasts. Limulus amoebocyte lysate assay and electromobility shift assay results revealed that chCATH-B1 bound to lipopolysaccharide (LPS) and lipoteichoic acid (LTA), which are the surface substances of the E. coli and S. aureus cell, respectively. A chemotactic assay results revealed that chCATH-B1 showed mouse-derived P-815 mastocytoma migrating activity dose-dependently but with a higher concentration, resulting in a loss of the activity. A semi-quantitative real-time RT-PCR analysis revealed that LPS stimulated chCATH-B1 gene expression in a dose-dependent manner and that the LPS-inducible chCATH-B1 gene expression was inhibited by the administration of dexamethasone. The chCATH-B1 mRNA levels in DT40 cells were also increased by the administration of bacterial LTA. The results indicate that bacterial toxins induce chCATH-B1 gene expression in the chicken BF and the peptide expressed in the organ would act against pathogenic microorganisms not only directly but also indirectly by attracting mast cells.
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Asuna
authors.initials: A
authors.lastname: Takeda
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsubaki
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Sagae
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Onda
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Inada
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Mochizuki
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Okumura
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan; Department of Biology, Faculty of Education and Integrated Arts and Sciences, Center for Advanced Biomedical Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjyuku-ku, Tokyo 162-8480, Japan.
authors.forename: Sakae
authors.initials: S
authors.lastname: Kikuyama
authors.affiliation: Department of Regulatory Biology, Faculty of Sciences, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. Electronic address: iwamuro@bio.sci.toho-u.ac.jp.
authors.forename: Shawichi
authors.initials: S
authors.lastname: Iwamuro
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D008223:Lymphoma / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: D023181:Antimicrobial Cationic Peptides; D001427:Bacterial Toxins; D012333:RNA, Messenger
keywords: Bursa of Fabricius; Cathelicidin; Chemotaxis; DT40 cells; Host defense peptides; Membrane destruction