大鼠實驗性心內膜炎模式中轉糖鏈球菌形成生物模之機制

Abstract

轉糖鏈球菌（Streptococcus mutans）主要存在於人體口腔中，是造成齲齒的主要病原菌。而當轉糖鏈球菌藉由口腔傷口進入血流，造成暫時性的菌血症，存在於血流中的細菌黏附到受損的心臟瓣膜上，形成贅疣（vegetation）引發感染性心內膜炎（infective endocarditis）。然而在以往的研究中，血小板在感染性心內膜炎中扮演甚麼角色並不清楚。本實驗發現在缺乏血小板的血漿中，細菌生物膜的形成會明顯被抑制，而當血小板存在時卻可以幫助細菌生物膜的形成。當外加入不同的血小板活化抑制劑也可以明顯觀察到細菌生物膜形成的減少，此外，當給與感染性心內膜炎大鼠抗凝血劑阿斯匹靈後，也可以有效干擾細菌體內生物膜的形成。這些結果暗示了血小板的活化對於細菌在血漿中形成生物膜是重要的。而進一步將血小板與細菌進行黏附，發現在人類免疫球蛋白存在下轉糖鏈球菌才能有效與血小板結合，顯示轉糖鏈球菌可能利用人類免疫球蛋白與血小板黏附並活化血小板有助其體內生物膜的形成。除了宿主因子與細菌的交互作用外，細菌本身是利用怎樣的調控機制也是值得研究，在本實驗室奇玄學長研究中發現一個response regulator的缺失可以影響轉糖鏈球菌體內生物膜的形成，而在microarray中也發現pspC 基因會明顯的被response regulator所調控。利用染色質免疫沉澱法與膠體電泳阻滯實驗也發現此response regulator可能直接調控pspC基因。而在pspC基因有所缺失的菌株中也可以明顯觀察到體外與體內生物膜形成的缺失。然而，測試多種細菌與宿主因子結合的實驗，發現野生株與PspC突變株與宿主因子結合並沒有明顯差異。顯示PspC在轉糖鏈球菌體內生物膜形成扮演重要角色，卻不影響轉糖鏈球菌與宿主因子的結合。

Streptococcus mutans is one of the principal causative agents of caries and an opportunistic pathogen of infective endocarditis (IE). Bacteremia is caused by S. mutans through the oral trauma and the circulating bacteria can adhere to the damaged valve to form a compact structure, vegetation (fibrin-platelet bacterial biofilm). However, the role of platelet is controversial in the pathogenesis of IE. In this study, plasma without platelets could inhibit the bacterial biofilm formation but enhance biofilm formation in the addition of platelets, which suggested platelets play an important role in the in vivo biofilm formation. After the addition of platelet activation inhibitors, the bacterial biofilm was reduced. When one of the platelet activation inhibitor, Aspirin, was applied to the IE rat model, the bacterial biofilm could be disturbed and lower bacterial load was obtained from the vegetation. These results suggest the activation of platelets is important in the pathogenesis of IE. To further dissect the interaction between platelets, plasma factors and bacteria, purified platelets are incubated with different plasma ingredients. Human immunoglobulin is discovered to essentially involve in the adherence of bacteria on the platelets. Except for the interaction of bacteria and platelets, the regulatory mechanism in the pathogenesis of IE is also studied. In our lab previous study, the defect in response regulator 11 can lead to the defect in IE biofilm formation and pspC gene is supposedly regulated by response regulator 11 in the microarray data. Chromatin immunoprecipitation and electrophoretic motility shift assay are performed in this study and further confirm response regulator 11 may directly regulate pspC gene. The deletion in pspC gene has defect in the in vitro and in vivo biofilm formation and this defect can be restored in the pspC gene complementation strain. However, no significant differences are obtained in the interaction of bacteria and host factors between PspC mutant and wildtype GS5. These results suggest the role of PspC protein may involve in the in vivo bacterial biofilm formation but not the interaction with host factors.