- 學位論文
酯解酵素基因可能參與金黃色葡萄球菌之生物膜形成
A Putative Esterase Might Be Involved in Biofilm Development of Staphylococcus aureus
摘要
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。
並列摘要
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.