- 學位論文
造成生物膜生成缺陷之金黃葡萄球菌突變株機制探討
Factors involved in a Staphylococcus aureus mutant strain with decreased biofilm activity
摘要
金黃色葡萄球菌(Staphylococcus aureus)為院內感染的主要致病菌之一，造成如導管、人工瓣膜的醫療器材感染等，且多與細菌在器材表面形成生物膜有關。生物膜為細菌的一種群體生活結構，其在生理特性與毒性等等皆與個體有不同的機制表現，導致更高的抗藥性及抵抗免疫的能力，造成治療上的困難。牽涉生物膜形成之相關因子非只有單一因子，在目前的研究中已經發現不少與金黃色葡萄球菌生物膜形成相關的基因，如ica、aap等；或是細菌的調控系統，如agr、sarA、σB等，會對不同的生物膜生成階段造成影響，然而仍未全盤瞭解生物膜生長機制。故本實驗室先前利用突變株庫的建立，篩選其中生物膜生成能力受影響之菌株，從中探討其造成缺陷的原因，以求對生物膜形成機轉更進一步認識。挑選其中一株生物膜生成能力明顯下降之菌株，經由野生株建構與其比較，發現突變株吸附表面的能力受到影響，但於動態生物膜生長並無與野生株不同。此突變株主要帶有putative esterase基因(est)的缺失，故利用API-ZYM套組測定突變株與野生株19種酵素活性，發現突變株脂解酵素能力略有上升。由於此段缺陷基因轉錄之蛋白功能及生理意義目前仍不清楚，無法明瞭其表型與此基因缺陷之關聯性，利用二維電泳及質譜進行蛋白質體分析，藉由鑑定兩株菌株間明顯差異表現之蛋白身份，以生物資訊學工具推測可能影響生物膜形成力路徑。另外亦建構缺陷基因多株抗體以求證實此段基因確實具有影響生物膜生成能力。
並列摘要
Staphylococcus aureus is one of pathogens with nosocomial infections. It causes many medical device-related infections which are associated with biofilm formation. These infections are hard to cure completely since biofilms are multicellular microorganisms, presenting different physiological characteristic and virulence, such as increased resistance to antibiotic and host defense. There are many genes which have been reported in involving biofilm formation, like ica, cap；or regulator systems, including agr, sarA, σ B, which influenced diverse phases of biofilm formation. However, many mechanisms are still remaining unclear.In a previous study, a transposon mutant library was generated and tested for the biofilm activity. One mutant strain with significantly lower activity of biofilm formation was obtained, with a putative esterase gene (est) disrupted by transposon.After constructed its wild type and compared with the mutant strain and complementary strains. There was no significant difference of all in flow phase of biofilm formation. Mutant strain had weaker attachment activity to form biofilm. There were two enzyme activities which were associated with lipase increased by using the API-ZYM kit to detected 19 enzyme activities.Due to the unknown function and biological significance of this disrupted gene, we used proteomic approach by 2D electrophoresis and mass spectrographic to search the proteins with different expression level compared to wild type to the mutant strain. Using bioinformatics tools, a possible pathway which may be correlated with biofilm activity was proposed. Furthermore, we constructed the Est poly-colonal antibody to verify the decreased biofilm phenotype cause of est gene disruption.