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

为了阐明一种口腔链球菌（streptococcus intermediusus， SI）对细胞溶素、中间溶素（cytolyin, intermediilysin， 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HepG2）的影响。由于在SI感染HepG2的TEM观察中发现SI细胞与人细胞表面微绒毛之间存在明显的结合，提示细胞内的细胞骨架网络参与了SI侵入人细胞的开始。实际上，随着SI感染的进展，细胞内肌动蛋白网络发生了变化。在对大量信号转导酪氨酸激酶的磷酸化分析中，通过低水平的ILY处理HepG2，证实了参与细胞骨架蛋白调控的酪氨酸激酶的磷酸化水平显著升高（是基础水平的5倍）。因此，该信号转导蛋白可能在依赖于ILY的人细胞特异性SI感染中发挥作用。此外，用PLA2抑制剂quinacrine处理后，SI感染引起的宿主细胞死亡倾向于减少。两个信号转导成员与SI感染的关系有待进一步研究。我们还检测了自噬标志物LC3的诱导，但在ST感染早期HepG2中未检测到LC3的显著升高。制备hud59转基因小鼠，研究体内SI感染机制。将含有小鼠CD59a启动子的huCD59基因构建体导入C57BL/6小鼠受精卵，经筛选获得2只转基因小鼠。正在从这些小鼠中制备两种+/+菌株。

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.