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Molecular mechanism of human-specific infection by an oral streptococcus, Streptococcusintermedius

口腔链球菌中间链球菌人类特异性感染的分子机制

基本信息

批准号：
18592004
负责人：
NAGAMUNE Hideaki
金额：
$ 2.57万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18592004/
关键词：
infectious disease bacteria oral streptococcus toxin intermedilysin

项目摘要

In order to clarify the human cell-specific infection depending on a cytolysin, intermedilysin (ILY) by an oral streptococcus Streptococcus intermedius (SI), the effects of ILY and SI cells on culture cells such as human hepatoma HepG2 were investigated. Because significant binding between SI cells and microvilli of human cell surface was found in TEM observation of SI-infected HepG2, some intracellular cytoskeletal network was suggested to participate in the start of SI invasion into human cells. Actually, it was revealed that intracellular actin network changed with proceeding of SI infection. It was confirmed that the phosphorylation level of a tyrosine kinase involved in regulation of cytoskeletal protein significantly increased(five-fold of basic level)by treatment of HepG2 with low level of ILY in phosphorylation analysis of a large number of signal transduction tyrosine kinases. So, this signal transduction protein was suggested to play a role in the human cell-specific SI infection depending on ILY. Moreover, host cell death induced by SI infection tended to decrease by the treatment with a PLA2 inhibitor, quinacrine. Further investigation is proceeding on the relationship among two signal transduction members and SI infection. Induction of LC3, a marker of autophagy, was also examined but remarkable increase of LC3 could not detect in HepG2 at the early stage of ST infection.Preparation of transgenic mice with huCD59 was carried out to investigate SI infection mechanism in vivo. A gene construct of huCD59 with mouse CD59a promoter was introduced into fertilized eggs of C57BL/6 mice and two transgenic mice were obtained after screening. Two+/+strains are preparing from these mice.

为了阐明口腔链球菌中间链球菌（SI）依赖于溶细胞素中间溶素（ILY）的人细胞特异性感染，研究了ILY和SI细胞对培养细胞如人肝癌HepG 2的影响。由于SI感染的HepG 2的TEM观察中发现SI细胞与人细胞表面的微绒毛之间存在显著的结合，因此提示细胞内的一些细胞骨架网络参与了SI侵入人细胞的开始。事实上，随着SI感染的进行，细胞内肌动蛋白网络发生了变化。在大量信号转导酪氨酸激酶的磷酸化分析中，通过用低水平的ILY处理HepG 2，证实了参与细胞骨架蛋白调节的酪氨酸激酶的磷酸化水平显著增加（基本水平的5倍）。因此，该信号传导蛋白被认为在依赖于ILY的人类细胞特异性SI感染中发挥作用。此外，SI感染诱导的宿主细胞死亡倾向于减少与PLA 2抑制剂，奎纳克林治疗。两个信号转导成员与SI感染的关系正在进一步研究中。同时检测了自噬标志物LC 3的诱导，但在ST感染的早期HepG 2中未检测到LC 3的显著增加。制备了huCD 59转基因小鼠，以研究SI感染的体内机制。将带有小鼠CD 59 a启动子的huCD 59基因构建体导入C57 BL/6小鼠受精卵中，筛选获得2只转基因小鼠。从这些小鼠中制备两种+/+品系。