臺灣博碩士論文加值系統

克雷白氏肺炎桿菌是一種伺機性病原菌，在台灣的糖尿病患者易引起肝膿瘍，但其原因尚不明瞭。克雷白氏肺炎桿菌具有多種致病因子，其中莢膜多醣體被視為是最重要的致病因子，我們在此利用小鼠感染試驗顯示從肝膿瘍病患分離出的菌株Kp 5具有高致病力，尿道感染菌株Kp 3則是低致病力，並利用小鼠腹腔吞噬細胞比較的吞噬程度，結果顯示Kp 3被吞噬的數量比Kp 5高。我們進一步利用ELISA技術分析細菌莢膜的lewis抗原，結果Kp 5有lewis b (Leb) 和sialyl-lewis x (sLex) 抗原表現，而Kp 3則無明顯lewis抗原表現。因此我們研究sLex抗原對血管內皮細胞的附著影響，已知受細胞激素活化的血管內皮細胞會表現可辨識sLex抗原的接受器，實驗結果顯示Kp 5對內皮細胞具有專一性的黏附作用。另外再利用sLex抗體抑制試驗，發現會降低Kp 5對活化內皮細胞的附著，說明Kp 5對內皮細胞的附著似乎與sLex有關。
另一研究方向是探討高致病菌株Kp 5本身的質體 pKP5具有何種生理意義。在定序結果中，得知質體pKP5為4,437 bp大小的ColE1類型質體，其所帶有的基因經過比對後，得到三個hypothetical proteins的ORFs和具plasmid entry exclusion功能的相似序列excl1基因、與RNA調節質體複製相關基因 (RNA I、RNA II、rom-like、oriV)、質體轉移的起始序列 (oriT) 以及可穩定質體的序列 (XerCD site-specific recombination sites kpr)等功能基因，目前只知pKP5的質體數約為8.5，且rom-like基因對質體數似乎沒有影響，並且利用質體不相容試驗得知pKP5可能延伸自於質體p15A。我們進一步將pKP5質體轉殖到肝膿瘍菌株Kp A6 (此菌株的PFGE基因型與Kp 5相似，但對小鼠為無致病力)，以及另外利用質體剔除比較pKP5的存在有無是否會影響Kp A6和Kp 5對小鼠的致病能力，結果顯示致病力均沒有明顯改變，推測pKP5應該與增加致病力無直接關聯。
綜合以上得知，sLex抗原可能會參與 Kp 5對內皮細胞的附著，而質體pKP5的明確功能仍然不明。

Klebsiella pneumonia (KP) is an opportunistic pathogen and can be found in diabetes mellitus patients with pyogenic liver abscess in Taiwan. The pathogenicity of this bacterium is not well understood. KP has many virulence factors, and the capsular polysaccharide (CPS) is believed to be an important virulence factor.
In this study, we found that the clinical isolate of Kp 5 strain from diabetic patients with liver abscess displayed high virulence in mice, however Kp 3 strain isolated from patients with urinary tract infection displayed low virulence. Mice peritoneal macrophages were used to investigate the phagocytosis of the bacteria. The results showed that Kp 3 was more easily phagocytized than Kp 5.
We used an enzyme-linked immunosorbent assay (ELISA) to detect the Lewis antigens in the KP capsule. The Kp 5 expressed significantly higher Leb and sLex than that of Kp 3. In addition, we investigated the role of sLex antigen in the adhesion of Kp 5 to human umbilical veins endothelial cell (HUVEC). The sLex antigen was known to be a ligand for the receptor expressed on cytokine-activated endothelial cells. Our data showed that Kp 5 can adhere to HUVEC (Multiplicity of infection<1, where Kp 3 can not). Furthermore, we were able to use sLex antibody to reduce the Kp 5 adhesion to HUVEC. These results suggest that sLex may play a role in Kp 5 adherence to HUVEC.
On the other hand, we had observed different plasmid profiles in the Kp3 and Kp5. The pKP5 plasmid from Kp 5 was purified and the DNA sequences were determined. The complete 4,437 bp sequence of the pKP5 plasmid showed homology to a ColE1-like plasmid and contained three ORFs for hypothetical proteins and a putative excl1 gene coded for plasmid entry exclusion function. The pKP5 plasmid seemed to replicate through a ColE1-like RNA-regulated mechanism (RNA I, RNA II, rom and oriV). The plasmid also contained a putative non-self-transmissible oriT and a homologous XerCD site-specific recombination site kpr for plasmid resolution and stabilization. In addition, we demonstrated that the pKP5 has copy number of 8.5 and the rom-like gene seems to have no distinct effect on plasmid copy number. The pKP5 may be derived from ColE1 type plasmid p15A by plasmid incompatibility experimentation.
Furthermore, we transformed the pKP5 plasmid to liver strain Kp A6 (a KP strain similar to Kp 5 by PFGE genotype, but not virulent in mice) and determined the virulence of the Kp A6 containing pKP5 plasmid in mice. In addition, we cured the pKP5 plasmid from Kp 5 and determined the virulence effect of the pKP5. Our results showed that there is no distinct difference of virulence in both experimentation, indicating the plasmid may not be related to mice virulence.
Taken together, we conclude that sLex antigen of Kp 5 may serve as an adhesion molecule to endothelial cell, and the function of pKP5 plasmid needs further studies.

指導教授對薦書
口試委員審定書
授權書.................................................. iii
致謝.................................................... iv
中文摘要................................................ v
英文摘要................................................ vi
目錄................................................... viii
圖表目錄................................................ x

壹、緒論
一、克雷白氏肺炎桿菌的簡介................................ 1
二、克雷白氏肺炎桿菌所引起的肝膿瘍......................... 1
三、克雷白氏肺炎桿菌的致病因子............................. 2
四、Lewis抗原在致病上之重要性............................. 4
五、質體對於致病影響..................................... 7
六、ColE1質體簡介....................................... 9
貳、研究動機............................................ 10
參、實驗材料方法........................................ 11
肆、 實驗結果
一、不同克雷白氏肺炎桿菌株感染小鼠致死率介.................. 21
二、利用腹腔週邊滲出細胞 (PEC)探討不同菌株的吞噬程度......... 21
三、E. coli 7E1 mucoid分析研究.......................... 21
四、質體p7E1分別在E. coli和Kp中對小鼠致病影響.............. 22
五、分析不同菌株莢膜的lewis抗原成份........................ 22
六、克雷白氏肺炎桿菌對內皮細胞的附著研究.................... 23
七、不同克雷白氏肺炎桿菌株的質體比較........................ 23
八、質體pKP5定序分析..................................... 24
九、質體pKP5轉殖菌株毒性試驗…….......................... 25
十、質體pKP5剔除以及對致病力影響.......................... 26
十一、質體pKP5質體數分析以及rom基因對質體數的影響........... 26
十二、質體之間的不相容性比較.............................. 27
伍、結果討論............................................ 28
陸、實驗圖表............................................ 36
柒、參考文獻............................................ 59

圖表目錄
圖一、克雷白氏桿菌相關的致病因子........................... 36
圖二、rcs基因組調節莢膜合成............................... 37
圖三、KP 3、5、95對小鼠致死率比較圖...................... 38
圖四、藉由誘導小鼠的腹腔週邊滲透細胞比較Kp 3、Kp 5的吞噬量... 39
圖五、利用PCR擴大篩檢E. coli 7E1的合成CPS基因組............ 40
圖六、以RT – PCR檢視CPS合成基因組中之一的gmd有無表現....... 42
圖七、比較質體p7E1分別在E. coli和KP中感染小鼠的致死率....... 43
圖八、以ELISA方式分析不同菌株的CPS之lewis抗原表現量......... 44
圖九、Kp3、Kp 5對內皮細胞的附著程度....................... 45
圖十、利用CsCl梯度離心純化方抽取Kp菌株的質體比較............ 46
圖十一、Kp 5之質體pKP5的定序分析與限制酶圖譜................ 47
圖十二、質體pKP5轉殖到Kp A6對小鼠致死影響.................. 48
圖十三、質體pKP5剔除對小鼠致死影響......................... 49
圖十四、pKP5質體數分析以及rom基因對質體數影響............... 50
圖十五、質體之間的質體不相容試驗與RNA I序列比對............. 51
圖十六、比較各個質體的Xer專一辨識重組位置序列............... 53
表一、本實驗所用到的引子.................................. 54
表二、Kp 5之質體pKP5的完整序列............................ 55
表三、質體pKP5上的ORF之位置及與NCBI比對結果................ 58

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