出版年:

研究生:

蔡佩珍

研究生(英文姓名):

Pei-Jane Tsai

論文名稱:

A群鏈球菌侵入上皮細胞之機轉:熱原性外毒素B之角色

英文論文名稱:

Pathogenic mechanism of group A streptococcus entry into epithelial cells: emphasis on the role of streptococcal pyrogenic exotoxin B

指導教授:

吳俊忠

指導教授(英文姓名):

Jiuun-Jong Wu

學位類別:

博士

校院名稱:

國立成功大學　

系所名稱:

基礎醫學研究所

學號:

S58841113

學年度:

87

語文別:

中文

論文頁數:

185

關鍵詞:

A 群鏈球菌 ; 蛋白水解 ; 熱原性外毒素B ; 侵入能力 ; 細胞凋亡

英文關鍵詞:

group A streptococcus ; protease ; streptococcal pyrogenic exotoxin B ;
invasion ; apoptosis

被引用次數:

0

[ 摘要 ]

A 群鏈球菌是引起人類疾病重要致命菌之一，自 1980 年以來，世界各地因 A 群鏈球菌感染而併發嚴重性的猩紅熱、風濕熱、毒性休克症候群、或壞死性肌膜炎之病例有逐漸增加的趨勢。但臺灣在過去幾年來，缺乏深入探討 A 群鏈球菌致病機轉的研究。為了更瞭解臺灣地區病例不斷增加及越趨嚴重的可能性，本研究收集近四年來南臺灣兩大醫院所分離之A群鏈球菌，發現在南臺灣 A 群鏈球菌株中 speA，speC，speF 分布的情形，與導致嚴重性 A 群鏈球菌感染的侵襲性菌株和非侵襲性菌株之間，並無明顯差異。然而 M1 protein 血清型及蛋白水解活性在侵襲性菌株和非侵襲性菌株，及在臨床症狀如: 軟組織壞死、休克及死亡有顯著的統計相關性。已有許多的證據顯示 A 群鏈球菌的蛋白水解，SPE B (cysteine protease)，在 A 群鏈球菌的感染中扮演許多相當重要的角色。為了更清楚看到蛋白水解與侵入能力之相關性，以上呼吸道之上皮細胞 (A-549 細胞株) 建立體外試驗模式，以穿透式電子顯微鏡方法，證實 A 群鏈球菌可吸附與侵入 A-549 細胞株，侵入細胞後並無法在短時間內快速複製生長。再以基因中斷法製備無蛋白水解之突變株與其野生株，分別感染上皮細胞，並外加純化之蛋白水解或其專一性的抑制劑，發現蛋白水解可能有增強 A 群鏈球菌侵入上皮細胞之能力。為了更瞭解 A 群鏈球菌感染機轉，觀測 A 群鏈球菌感染上皮細胞後所發生的變化，以蛋白水解之野生株與其突變株感染上皮細胞後，發現隨著時間的增長，細菌毒殺細胞的能力亦愈加強；同時，其細胞形態也發生變化：細胞核濃染及 apoptotic body 的產生；另外，亦發現有 DNA fragmentation 的現象，此皆為細胞凋亡 (apoptosis) 的明顯特徵；同時以細胞流體分析儀分析凋亡細胞的比例，發現死菌並不能導致細胞的死亡，而且以細胞骨架抑制劑 cytochalasin D 處理，亦發現細菌必須要能侵入細胞內才能造成細胞自殺性死亡。此外，也發現具蛋白水解的菌株可引發較大量的凋亡細胞。藉由細胞 caspase 抑制劑，得知 A 群鏈球菌感染上皮細胞後，可經由 caspase 的途徑造成細胞自戕，而且蛋白水解的存在可能有增強 A 群鏈球菌造成細胞凋亡的能力。因此，綜合本論文的研究，A 群鏈球菌的感染，不僅可進入上皮細胞中並引發 caspase 的途徑造成細胞自戕外，蛋白水解的更會影響感染過程的嚴重性。這些研究結果不但提供了一些 A 群鏈球菌與上皮細胞相互作用的訊息，同時也為 A 群鏈球菌感染之機轉開啟了另一種思考方向。

[ 英文摘要 ]

Streptococcus pyogenes, group A streptococcus (GAS), is the major causative agent of a number of human diseases. Since 1980, striking increase in the frequency and severity of streptococcal infections including soft tissue necrosis, streptococcal toxic shock-like syndrome (STSS), and scarlet fever is worldwide. However, there were only a few reports in the pathogenesis of GAS in Taiwan. To determine whether the spectrum of invasive GAS diseases in Taiwan parallels the increasing incidence and severity seen in other countries, we conducted a 4-year study reviewing all invasive GAS infections at two hospital centers in southern Taiwan. Neither the presence of speA, speC, or speF of the isolates is implicated in any particular clinical syndrome of patients with invasive GAS diseases. High protease activity and M1 serotype of isolates were significantly associated with clinical signs of STSS and mortality. The M1 serotype and protease activity, as well as the host immunity status may play significant roles in the pathogenesis of invasive diseases in Taiwan. Several lines of evidence suggest that streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, plays an important role in GAS infection. In order to understand the role of protease in the pathogenesis of streptococcal infection and the relationship between invasion and protease, I have established an internalization assay on the basis of resistance of intracellular streptococci to penicillin and the internalization was confirmed by the electron microscope. Isogenic protease mutants were constructed by using integrational plasmid to disrupt the speB gene. The mutants had growth rates similar to those of the wild-type strains. When invading A-549 cells, the mutants had a two- to threefold decrease in invasion activity compared to that of the wild-type strains. The invasion activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect adhesive ability of GAS. These results suggest that streptococcal cysteine protease can enhance the invasion of GAS into human respiratory epithelial cells. The fate of the GAS-infected epithelial cells was further studied. GAS has the ability to invade A-549 and HEp-2 cells. Both A-549 and HEp-2 cells were killed by infection with GAS. Epithelial cell death mediated by GAS at least in part through apoptosis, as shown by cellular morphology, staining with propidium iodide, and DNA fragmentaion. Apoptosis can be blocked by using cytochalasin D which interferes with cytoskeleton function. The caspase inhibitors, Z-VAD.CMK, Ac-YVAD.FMK, and Ac-DEVD.FMK, inhibited GAS-induced apoptosis. I further examined whether SPE B is involved in the induction of apoptosis. The speB isogenic mutants had less ability to induce cell death than those of the wild-type strains. When the A-549cells were cocultured with the mutant and SPE B for 2 h, the number of intracellular bacteria increased to the level of wild-type strains, whereas, the percentage of apoptotic cells did not increase. These results demonstrate for the first time that GAS induces apoptosis of epithelial cells and internalization is required for apoptosis. Th caspase pathway is involved in GAS-induced apoptosis, and expression of SPE B within the cells enhances apoptosis. This study provides a novel information regarding GAS interacting with the epithelial cells, and opens a new spectrum to study the pathogenic mechanism of GAS.