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Functional Analysis of Antimicrobial Peptides in Protection from Bacterial Infection

抗菌肽预防细菌感染的功能分析

基本信息

批准号：
15590406
负责人：
KUWANO Koichi
金额：
$ 1.86万
依托单位：
Kurume University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590406/
关键词：
antimicrobial peptide Mycoplasma pneumoniae β-defensin CRAMP E.coli

项目摘要

Initially, we examined the role of inducible hBD in the acute phase of in vitro M.pneumoniae infection. We observed that chemically synthesized hBD-2 and hBD-3, but not hBD-1, show effective antimicrobial activity against M.pneumoniae. To induce hBD production, a human pulmonary squamous cell line EBC-1 was incubated with a potent stimulant, IL-1β. Culture supernatant and total RNA from the EBC-1 were examined for hBD expression. hBD-2 in the supernatant was detected by western blot analysis. In addition, hBD-2 mRNA was strongly up-regulated by IL-1bβ, but neither hBD-1 nor hBD-3 was apparently up-regulated. To determine whether hBD induces the protection from infection, IL-1β-treated EBC-1 cell were infected with M.pneumoniae. IL-1β-treated EBC-1 significantly inhibited the growth of M.pneumoniae, as judged by colony assay. Thus, our results suggest that hBD-2 produced by IL-1β-treated EBC-1 cells plays a role in the protection of early stage of M.pneumoniae infection.Subsequently, roles of antimicrobial peptides, in particular CRAMP, in in vivo infection model were examined. There seems to be no reports regarding the CRAMP and Mycoplasma infection. CRAMP, a family of cathelicidin, was chemically synthesized. The CRAMP showed an effective antimicrobial activity against M.pneumoniae. To induce CRAMP in vivo, BALB/c mice were intranasally infected with M.pneumoniae, and infected lungs taken from the mice at 6,12,24 and 48 hours postinfection were examined for CRAMP expression. Western blot analysis showed that the homogenate from the lungs at 24 hours after infection contains CRAMP. In addition, CRAMP mRNA from the infected lungs was detected by using RT-PCR. Immunostaining showed that there are many CRAMP-positive neutrophils in the peribronchial area, but no definitive CRAMP-positive epithelial cells. At present underway is investigation on the association between CRAMP-positive neutrophils and protective effect

最初，我们研究了诱导型hBD在体外肺炎支原体感染急性期的作用。我们观察到，化学合成的hBD-2和hBD-3，但不是hBD-1，对肺炎支原体显示有效的抗菌活性。为了诱导hBD产生，将人肺鳞状细胞系EBC-1与强效刺激剂IL-1β孵育。检测培养物上清液和来自EBC-1的总RNA的hBD表达。Western blot检测上清中hBD-2的表达。此外，IL-1bβ强烈上调hBD-2 mRNA，但hBD-1和hBD-3均未明显上调。为了确定hBD是否诱导对感染的保护，用肺炎支原体感染IL-1β处理的EBC-1细胞。经IL-1β处理的EBC-1可显著抑制肺炎支原体的生长。因此，IL-1β诱导EBC-1细胞产生的hBD-2在肺炎支原体感染的早期起保护作用。似乎没有关于CRAMP和支原体感染的报告。化学合成了抗菌肽家族CRAMP。CRAMP对肺炎支原体具有有效的抗菌活性。为了在体内诱导CRAMP，BALB/c小鼠鼻内感染肺炎支原体，并在感染后6、12、24和48小时从小鼠中取出感染的肺检测CRAMP表达。Western印迹分析显示，感染后24小时的肺匀浆含有CRAMP。RT-PCR检测感染肺组织中CRAMP mRNA的表达。免疫组化显示支气管周围有许多CRAMP阳性中性粒细胞，但没有明确的CRAMP阳性上皮细胞。目前正在研究CRAMP阳性中性粒细胞与保护作用之间的关系