A Putative Esterase Might Be Involved in Biofilm Development of Staphylococcus aureus

金黃色葡萄球菌 (Staphylococcus aureus ) 是人類重要的致病菌，可引起多種臨床症狀如傷口感染、食物中毒，及嚴重的骨髓炎、菌血症、心內膜炎等。臨床上可發現金黃色葡萄球菌常以生物膜的型態引起疾病，特別在醫療器材如導管、人工瓣膜等相關的感染上。生物膜是細菌吸附於表面，被胞外基質包覆所組成的多細胞群體結構，處於生物膜的型態有利於細菌逃避宿主的免疫系統，及抵抗環境中各種壓力，包括增加細菌的抗藥性，導致生物膜的感染在治療上非常困難，而使生物膜相關的研究逐漸受到重視。 本論文中利用跳躍基因致突變法 (transposon mutagenesis) 建構突變株庫，以生物膜微量培養盤試驗，從中篩選到一株生物膜形成能力明顯降低的突變株，並作進一步的分析，希望藉此找出金黃色葡萄球菌中可能與生物膜形成相關的基因，並探討此基因參與生物膜形成的機制。此突變株先經Southern blot確認transposon 為單一嵌入其基因體中，再以反向PCR及 DNA定序找出 transopson所在位置，並與S. aureus NCTC8325全基因體序列做比對分析，結果發現被破壞的區域為一未知功能的基因，具有 α/β hydrolase的蛋白質功能區，預測的產物為酯解酵素。 比較突變株和野生型菌株的生長曲線，發現兩者在生長速率上無明顯的差異，顯示生物膜形成缺陷並非由於生長慢使菌體累積不足造成。進行初級吸附的實驗，突變株吸附至聚苯乙烯表面的能力比野生型菌株差；分析菌體的疏水性質，突變株的疏水性較野生型菌株低，結果顯示突變株在菌體吸附於表面的階段上具有缺陷。 透過基因補償實驗及建構基因刪除突變株，討論生物膜形成的缺陷是否因為此基因被破壞後造成的影響。並嘗試利用一維及二維電泳比較突變株與野生型菌株在蛋白質表現上的差異，找出可能與生物膜形成相關的蛋白質。Staphylococcus aureus is an important human pathogen, which can cause a variety of diseases, for example, wound infection, food poison, life-threatening osteomyelitis, bacteremia and endocartidis. Biofilm infections associated with S. aureus account for serious clinical problems, especially in medical device-related infections. Biofilms are multi-cellular communities consist of surface-attached microorganisms encased by extracellular matrix. Bacteria living in biofilm mode can escape from host immune systems and resist to adverse stress in the environment. Biofilm cells are more tolerant to antimicrobial agents thus are notoriously difficult to treat. In this study, a transposon mutant library was generated by transposon mutagenesis and the biofilm activity was screened by microtiter plate assay. One mutant strain with significantly reduced biofilm capacity has been selected. Further analysis was performed to identity the transposon disrupted gene and it’s role in S. aureus biofilm formation. Single transposon insertion was confirmed by Southern blot analysis. Comparison with S. aureus NCTC8325 whole genome sequence, the region disrupted by transposon encodes a putative esterase, which belongs to the α/β hydrolase family. There was no significant difference in growth rate between wild type and the mutant, suggest that the reduced biofilm phenotype was not due to insufficient cell accumulation. In the initial attachment experiment, the mutant displayed weaker adherence to polystyrene surface than wild type. The mutant strain also exhibited lower hydrophobicity compared to wild type. These results implied that the surface attachment capacity of the mutant was impaired. Gene complementation and deletion experiments were conducted to verify the altered biofilm phenotype resulted from est gene disruption. We used 2D electrophoresis to compare the protein expression profile between wild type and mutant, and attempted to identify proteins possibly involved in biofilm formation.口試委員會審定書 i謝 ii文摘要 iiibstract v 錄 vii一章 緒論 1二章 材料與方法 9.1 實驗用菌株及質體 9.2 實驗所用之引子 10.3 建構突變株庫 (mutant library) 11.4 生物膜微量盤試驗 (biofilm microtiter plate assay) 12.5 金黃色葡萄球菌染色體核酸 (chromosomal DNA) 之萃取 13.6 南方墨點法 (Southern blot) 14.7 反向聚合酵素連鎖反應 (inverse PCR) 16.8 核酸定序 (DNA sequencing) 17.9 測定生長曲線 18.10 初級吸附 (initial adherence) 能力測試 19.11 菌體疏水性 (hydrophobicity) 測試 19.12 蛋白質一維電泳分析 20.13 Wall teichoic acid (WTA) 萃取及電泳分析 21.14 蛋白質二維電泳分析 23.15 測定est基因之表現 24.16 尋找est 基因之轉錄起點 (transcription start site ) 27.17 基因補償實驗 (complementation) 29.18 建構基因刪除突變株 (deletion mutant) 30三章 實驗結果 32.1 建構突變株庫 32.2 生物膜微量培養盤試驗 ( biofilm microtiter plate assay ) 32.3 Southern blot 確認 transposon insertion 33.4 反向聚合酵素連鎖反應 (inverse PCR) 33.5 序列比對結果 34.6 測定生長曲線 35.7 初級吸附能力 35.8 菌體疏水性 35.9 蛋白質一維電泳分析 36.10 WTA 分析 36.11 蛋白質二維電泳分析 36.12 est基因表現 37.13 est 基因轉錄起點 38.14 基因補償實驗 (complementation) 38四章 討論 39.1 突變株庫代表性 39.2 突變株之篩選方式 39.3 Mutant_1基因體與wild type之差異 40.4 探討mutant_1生物膜缺陷的發生階段 40.5 est基因序列分析 42.6 基因補償株 (complemented strain) 分析 43.7 二維電泳 44.8 結論與未來方向 45五章 實驗之附圖 46 一. 生物膜微量培養盤試驗 (biofilm microtiter plate assay) 結果 46 二. 96孔盤底部細胞吸附情形 (400X 放大) 47 三. 生物膜累積 (biofilm accumulation) 實驗結果 48 四. Southern blot：以ermB probe 確認Tn551 transposion 49 五. Inverse PCR示意圖及電泳結果 50 六. 生長曲線 51 七. 初級吸附能力比較 52 八. 菌體疏水性測試結果 53 九. SDS-PAGE分析whole cell extract及 surface protein 54 十. Wall teichoic acid (WTA) 分析. 54 十一. 利用二維電泳比較wild type及mutant_1蛋白質表現之差異 57 十二. 以RT-PCR偵測est基因之mRNA表現 58 十三. est 基因上游DNA序列分析 59 十四. est 基因 PCR產物電泳圖 60 十五. 以est probe 進行Southern blot 偵測周圍DNA序列結構 61 十六. est基因周圍1 Kb區域ORF組成情形 61 十七. 基因補償株之生物膜形成能力與蛋白質分析 62考文獻 6