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研究生:辛祈諠
研究生(外文):Hsin Chi-Hsuan
論文名稱:探討調控肺炎克雷白氏桿菌莢膜多醣體表現的毒力因子rmpA及rmpA2
論文名稱(外文):Roles of rmpA and rmpA2 to regulate capsular polysaccharide synthesis and virulence of Klebsiella pneumoniae
指導教授:蕭樑基蕭樑基引用關係林永崇林永崇引用關係
指導教授(外文):Siu, Leung-KeiLin, Jung-Chung
口試委員:林靖婷陳政男陳盈璁
口試委員(外文):Lin,Ching-TingChen,Chang-NanChen,Ying-Tsong
口試日期:2012-06-28
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:100
語文別:中文
論文頁數:82
中文關鍵詞:肺炎克雷白氏桿菌毒力因子
外文關鍵詞:Klebsiella pneumoniaermpA and rmpA2
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肺炎克雷白氏桿菌為伺機性感染病原菌,常造成的疾病包括肺炎,尿道感染,菌血症。肺炎克雷白氏桿菌常表現大量的莢膜多醣體,使菌體外觀呈現黏稠狀,做為細菌產生抗白血球吞噬及抗補體毒殺的毒力因子。
之前的研究提出rmpA及rmpA2基因為肺炎克雷白氏桿菌中能夠與莢膜多醣體的基因啟動子結合,做為活化莢膜多醣體合成的轉錄因子。此外,發現肺炎克雷白氏桿菌的質體可同時帶有rmpA及rmpA2基因,而實驗室之前的研究發現臨床上的肺炎克雷白氏桿菌同時帶有rmpA及rmpA2基因高達80%,在基因序列分析上rmpA與rmpA2具有81%的相似性。
本篇研究目的是釐清rmpA及rmpA2基因與調控莢膜多醣體的相關性及對細菌毒力的影響。首先利用pACYC184的質體建構分別帶有rmpA、rmpA2、rmpA/rmpA2基因的三個質體並轉型至HK30(K2)及DM34(K34)不帶有任何rmpA基因型的肺炎克雷白氏桿菌。利用莢膜多醣體定量分析及血清毒殺、及白血球吞噬試驗,評估哪種組合對於調控莢膜多醣體的表現及影響細菌毒力較為重要。結果顯示rmpA/rmpA2比起單獨帶有rmpA及rmpA2菌株表現更多莢膜多醣體,rmpA及rmpA2存在時細菌抗吞噬能力增強,但以小鼠腹膜炎模式評估毒力時發現HK30及DM34帶有rmpA、rmpA2、rmpA/rmpA2毒力表現皆與野生株相似。
進一步評估rmpA及rmpA2的功能,另外建構一支NVT2001S(K2)菌株將其個別剔除rmpA及rmpA2基因及同時剔除rmpA/rmpA2基因,以毒力試驗評估其功能,結果顯示在剔除rmpA及rmpA/rmpA2使莢膜多醣體表現量降低及抗嗜中性球吞噬能力下降,並在老鼠腹膜炎模式中使細菌毒力下降,而在剔除rmpA2後進行上述的毒力試驗與野生株比較後並無顯著差異。
本篇研究證實同時帶有rmpA/rmpA2基因的菌株使莢膜多醣體表現量增加;rmpA作為調控肺炎克雷白氏桿菌莢膜多醣體合成及影響細菌毒力的的基因,而缺陷的rmpA2並無法調控肺炎克雷白氏桿菌莢膜多醣體合成及影響細菌毒力。

Klebsiella pneumoniae is an opportunistic pathogen which can cause pneumonia, urinary tract infection and bacteremia as well as liver abscess. K. pneumoniae usually has an encapsulated capsular polysaccharide (CPS), which exhibit a hyper-mucoid phenotype. CPS is usually responsible for resistance to phagocytosis or serum killing by bactericidal factors.
Previous studies have demonstrated that rmpA (regulate of mucoid phenotype A) and rmpA2 both encode a transcriptional activator for the cps expression by binding directly to the putative promoters. Our investigation showed that the clinical strains usually have both rmpA and rmpA2 (80%), while rmpA2 has a high degree of DNA sequence similarity (81%) in comparison with rmpA.
The aim of this study was to investigate the association between rmpA and rmpA2 in the regulation of CPS synthesis and the effects of these two genes on the virulence of K. pneumoniae. Three different plasmids containing rmpA, rmpA2, or rmpA/rmpA2 have been constructed, respectively, and then were transformed into clinical isolates of K. pneumonaie. HK30 (serotype K2) and DM34 (serotype K34), that both rmpA and rmpA2 are undetected. The roles of rmpA and rmpA2 were assessed by CPS quantification, serum resistance, neutrophil phagocytosis, and mouse lethality assay. Our results showed that K. pneumoniae strains, HK30 and DM34, with plasmid containing both of rmpA and rmpA2 have the highest production of CPS than the other strains carrying only rmpA or rmpA2 plasmid. In neutrophil phagocytosis assay, our results found both rmpA and rmpA2 could enhance the ability of anti-phgocytosis in K. pneumoniae. However, no significant difference of virulence has been found between strains with plasmid containing rmpA and/or rmpA2 and the parental strain in a mouse peritonitis model.
To further evaluate the role of rmpA and rmpA2, three mutants, the single deletion of rmpA or rmpA2 (ΔrmpA and ΔrmpA2) and the double deletion of rmpA and rmpA2 (ΔrmpA/rmpA2), were constructed from K. pneumoniae NVT2001S. In contrast to the parental strain, ΔrmpA and ΔrmpA/rmpA2 mutants both showed a reduced expression of CPS, a lower anti-phagocytosis ability, and a lower virulence in a mouse peritonitis model, while ΔrmpA2 mutant presented similar results on these assays.
In summary, our results suggest that rmpA play an important role than the defective rmpA2 in CPS synthesis and virulence in K. pneumoniae.

第一章 前言 1
第一節 肺炎克雷白氏桿菌之特性 2
第二節 調控肺炎克雷白氏桿菌莢膜多醣體合成的rmpA及rmpA2基因 5
第三節 rmpA與rmpA2與其它基因之交互作用 7
第四節 肺炎克雷白氏桿菌抗是中性球吞噬及抗血清作用 8
第五節 本研究假設及目標 9
第二章 材料與方法 11
一、材料 12
第一節 菌株 12
第二節 質體 12
第三節 引子 12
第四節 試藥 13
第五節 儀器 16
第六節 試劑配方 16
第七節 培養基配方 17
二、實驗方法 20
第一節 聚合酶連鎖反應 20
第二節 莢膜血清型之分型 21
第三節 質體DNA的抽取 22
第四節 勝任細胞之製備 22
第五節 rmpA基因轉殖 23
第六節 肺炎克雷白氏桿菌基因之轉型. 23
第七節 建構NVT2001S之突變株. 24
第八節 RNA之抽取. 25
第九節 cDNA之製備. 26
第十節 cDNA聚合酶連鎖反應. 26
第十一節 肺炎克雷白氏桿菌莢膜定量 27
第十二節 血清補體毒殺試驗 28
第十三節 嗜中性球吞噬反應 29
第十四節 小鼠腹膜炎模式 33
第十五節 統計分析 34
第三章 實驗結果 35
第一節 K1/K2及非K1/K2血清型菌株之rmpA基因分型 36
第二節 rmpA與rmpA2基因序列挑選 37
第三節 建構rmpA、rmpA2、rmpA/rmpA2質體 37
第四節 定量莢膜多醣體表現 38
第五節 菌株的血清毒殺率 38
第六節 菌株被嗜中性球吞噬比率 39
第七節 菌株小鼠致死率 40
第八節 NVT2001S及突變株細菌毒力表現 40
第四章 討論 43
第一節 rmpA與rmpA2基因在肺炎克雷白氏桿菌中扮演的角色 44
第二節 rmpA與rmpA2調控莢膜多醣體合成 45
第三節 rmpA與rmpA2基因與細菌毒力的關係 46
第四節 rmpA與rmpA2與肺炎雷白氏桿菌血清型的關係 47
第五節 rmpA基因在肺炎雷白氏桿菌中的重要性 48
第五章 結論 50
第六章 參考文獻 74

第一章 前言 1
第一節 肺炎克雷白氏桿菌之特性 2
第二節 調控肺炎克雷白氏桿菌莢膜多醣體合成的rmpA及rmpA2基因 5
第三節 rmpA與rmpA2與其它基因之交互作用 7
第四節 肺炎克雷白氏桿菌抗是中性球吞噬及抗血清作用 8
第五節 本研究假設及目標 9
第二章 材料與方法 11
一、材料 12
第一節 菌株 12
第二節 質體 12
第三節 引子 12
第四節 試藥 13
第五節 儀器 16
第六節 試劑配方 16
第七節 培養基配方 17
二、實驗方法 20
第一節 聚合酶連鎖反應 20
第二節 莢膜血清型之分型 21
第三節 質體DNA的抽取 22
第四節 勝任細胞之製備 22
第五節 rmpA基因轉殖 23
第六節 肺炎克雷白氏桿菌基因之轉型. 23
第七節 建構NVT2001S之突變株. 24
第八節 RNA之抽取. 25
第九節 cDNA之製備. 26
第十節 cDNA聚合酶連鎖反應. 26
第十一節 肺炎克雷白氏桿菌莢膜定量 27
第十二節 血清補體毒殺試驗 28
第十三節 嗜中性球吞噬反應 29
第十四節 小鼠腹膜炎模式 33
第十五節 統計分析 34
第三章 實驗結果 35
第一節 K1/K2及非K1/K2血清型菌株之rmpA基因分型 36
第二節 rmpA與rmpA2基因序列挑選 37
第三節 建構rmpA、rmpA2、rmpA/rmpA2質體 37
第四節 定量莢膜多醣體表現 38
第五節 菌株的血清毒殺率 38
第六節 菌株被嗜中性球吞噬比率 39
第七節 菌株小鼠致死率 40
第八節 NVT2001S及突變株細菌毒力表現 40
第四章 討論 43
第一節 rmpA與rmpA2基因在肺炎克雷白氏桿菌中扮演的角色 44
第二節 rmpA與rmpA2調控莢膜多醣體合成 45
第三節 rmpA與rmpA2基因與細菌毒力的關係 46
第四節 rmpA與rmpA2與肺炎雷白氏桿菌血清型的關係 47
第五節 rmpA基因在肺炎雷白氏桿菌中的重要性 48
第五章 結論 50
第六章 參考文獻 74


表目錄

表1 實驗菌株與質體 52
表2 實驗用引子 53
表3 本實驗挑選之臨床菌株 54
表4 K1/K2血清型中rmpA基因型分布率 58
表5 非K1/K2血清型中rmpA基因型分布率 59
表6 臨床上rmpA2基因序列分析 60
表7 rmpA基因與血清毒殺之關聯性 61
表8 剔除rmpA及rmpA2基因與血清毒殺之關聯性 62



圖目錄
圖1 rmpA、rmpA2及rmpA/rmpA2基因之轉殖 63
圖2 HK30及DM34野生株與突變株莢膜多醣體表現量 65
圖3 HK30與DM34野生株與突變株被嗜中性球吞噬比率 66
圖4 HK30野生型菌株與突變株小鼠存活率 67
圖5 DM34野生型菌株與突變株小鼠存活率 68
圖6 NVT2001S突變株即時核糖核酸定量試驗 69
圖7 NVT2001S野生型菌株與突變株莢膜多醣體表現量 70
圖8 NVT2001S野生型菌株與突變株抗白血球吞噬比率 71
圖9 NVT2001S野生型菌株與突變株小鼠存活率 72



附錄目錄
附圖1 肺炎克雷白氏桿菌之毒力因子………………………………73


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