Takeda Asuna, Tsubaki Takashi, Sagae Nozomi, Onda Yumiko, Inada Yuri, Mochizuki Takuya, Okumura Kazuo, Kikuyama Sakae, Kobayashi Tetsuya, Iwamuro Shawichi
Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
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.
Peptides. 2014 Sep;59:94-102. doi: 10.1016/j.peptides.2014.06.012. Epub 2014 Jun 28.
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.
鸡cathelicidin-B1（chCATH-B1）是鸡法氏囊（BF）的一种主要宿主防御肽。为了研究chCATH-B1基因在BF中表达的机制，我们聚焦于源自鸡法氏囊淋巴瘤的DT40细胞系作为分析模型。从DT40细胞中克隆出编码chCATH-B1前体的cDNA。该cDNA的核苷酸序列与BF的chCATH-B1相同。肉汤稀释分析表明，合成的chCATH-B1对大肠杆菌和金黄色葡萄球菌均表现出显著的防御活性。扫描显微镜分析表明，chCATH-B1通过形成泡和原生质球破坏细胞膜来抑制细菌生长。鲎试剂检测和电泳迁移率变动分析结果显示，chCATH-B1分别与大肠杆菌和金黄色葡萄球菌细胞的表面物质脂多糖（LPS）和脂磷壁酸（LTA）结合。趋化分析结果显示，chCATH-B1剂量依赖性地表现出对小鼠来源的P-815肥大细胞瘤的迁移活性，但在较高浓度时活性丧失。半定量实时RT-PCR分析表明，LPS以剂量依赖性方式刺激chCATH-B1基因表达，且地塞米松给药可抑制LPS诱导的chCATH-B1基因表达。细菌LTA给药也可使DT40细胞中的chCATH-B1 mRNA水平升高。结果表明，细菌毒素可诱导鸡BF中chCATH-B1基因表达，且该器官中表达的肽不仅可直接作用于病原微生物，还可通过吸引肥大细胞间接发挥作用。
