臺灣博碩士論文加值系統

克雷伯氏肺炎桿菌是引發台灣化膿性肝膿瘍 ( pyogenic liver abscess,
PLA ) 的最主要病原體，而糖尿病病患是罹患 PLA 的高風險族群之一。
然而，高血糖提高克雷伯氏肺炎桿菌引起 PLA 的風險之機制目前仍然不
清楚。生物膜 ( biofilm ) 的生成與細菌致病有關，生物膜可以維持細菌的
生存並且能夠隔絕外界有害物質的攻擊像是抗生素的作用。細菌的附著是
生物膜形成的第一步驟，因此，細菌的黏附能力是生物膜形成的重要因素
之一，其中纖毛 ( fimbriae ) 在這步驟扮演了一個重要的角色。目前克雷
伯氏肺炎桿菌具有兩型纖毛，分別為第一型與第三型纖毛，而第三型纖毛
又為克雷伯氏肺炎桿菌所特有的，第三型纖毛主要是由 mrk 基因叢 ( gene
cluster ) 所調控，是克雷伯氏肺炎桿菌的重要致病因子之一。文獻指出，
在很多腸內桿菌科細菌中，cAMP 受體蛋白 ( cyclic AMP receptor protein,
CRP ) 調控許多細菌毒力基因的表達，包括纖毛和生物膜形成。根據本實
驗室先前的實驗結果發現，mrkA ( 第三型纖毛的主要次單元 ) 的啟動子
區域有預測到 CRP 的結合位，進一步也證實了 CRP 確實會結合在 mrkA
的啟動子上。因此，本篇論文著重於探討 CRP 與克雷伯氏肺炎桿菌第三
型纖毛之間的調控關係。本研究嘗試許多基因剔除的方法，最後也成功獲
得了 crp 突變株，也進一步證實了 CRP 確實扮演著調控 MrkA 的角
色。
此外，先前實驗室研究也發現到克雷伯氏肺炎桿菌第三型纖毛的表現
量會受到葡萄糖濃度所調控，然而，當大量表達 KPN_00353- 00351 ( EIIA,
EIIB, EIIC homolog ) 後，則不會受到葡萄糖所調控。因此，本篇論文也進
一步探討 KPN_00353-00351 與第三型纖毛之間的調控關係，也證實了
KPN_00353-00351 調控 MrkA 的表現是必須依賴 CRP 的調控途徑。

Klebsiella pneumoniae, is the predominant pathogen isolated from
pyogenic liver abscess (PLA) of diabetic patients in Taiwan. However, the
effect of high blood glucose associated with K. pneumoniae strains remain
largely unknown. Bacterial biofilm represents a key virulence determinant in
promoting bacterial persistence and resistance to antibiotics. The ability of
bacterial adherence to biotic or abiotic substrate is an essential step for biofilm
formation. Such adherence can be mediated by bacterial fimbriae, which are
also important virulence factors in many bacteria. Type 3 fimbriae, encoded by
the mrkABCDF operon genes, are important virulence factors in K. pneumoniae
pathogenesis. In enterobacteria, cyclic AMP (cAMP) receptor protein (CRP), as
a global regulator, plays a vital role in numerous phenotypes. CRP protein
regulates several essential bacterial virulence gene expressions, including
fimbriae and biofilm formation, responsing to intracellular concentration of
cAMP.
In our preliminary study, we found that CRP can exactly bind to the
putative CRP binding site which located at the promoter region of mrkA
(encoding type 3 fimbriae subunit) in K. pneumoniae. Thus, our purpose is to
clarify the effect of cAMP receptor protein (CRP) on type 3 fimbriae in K.
pneumoniae. Loss-of-function mutagenesis is an important tool to characterize
gene functions. Here, we used several knockout systems to get crp mutants.
Finally, we successfully got crp mutants in K. pneumoniae MGH78578 by
homologous recombination (HR) using our modified pK18mobsacB suicide
plasmid. We found that active form of CRP can directly regulate MrkA
expression in K. pneumoniae. Moreover, previous data also showed that
different glucose level can regulate the expression of type 3 fimbriae. However,
the effect of glucose will be counteracted as we overexpressed KPN_00353-
00351 in K. pneumoniae. Thus, we also want to clarify the effect of
KPN_00353-00351 on type 3 fimbriae in K. pneumoniae. In this study, our data
indicated that the regulation of MrkA expression by KPN_00353-00351 is also
dependent on the CRP pathway.

中文摘要 .............................................................................................................. I
Abstract ............................................................................................................... II
目錄 ................................................................................................................... III
附圖表目錄 ....................................................................................................... VI
第一章、前言 ..................................................................................................... 1
1.1 克雷伯氏肺炎桿菌 ( Klebsiella pneumoniae ) 簡介 ........................ 1
1.2 肝膿瘍 ( liver abscess ) 簡介 ............................................................. 3
1.3 克雷伯氏肺炎桿菌之化膿性肝膿瘍 ( Klebsiella pneumoniae liver
abscess, KPLA ) .............................................................................................. 3
1.4 克雷伯氏肺炎桿菌致病因子 .............................................................. 4
1.5 克雷伯氏肺炎桿菌生物膜的形成 ...................................................... 6
1.6 克雷伯氏肺炎桿菌纖毛 ...................................................................... 9
1.7 磷酸轉移酶系統 ( Phosphotransferase system, PTS system ) ......... 10
1.8 cAMP 受體蛋白 ( Cyclic AMP receptor protein, CRP ) .................. 13
1.9 研究目的 ............................................................................................ 14
第二章、實驗材料 ........................................................................................... 18
2.1 實驗使用菌株 .................................................................................... 18
2.2 實驗使用質體 .................................................................................... 19
2.3 限制酶 ................................................................................................ 21
2.4 抗生素 ................................................................................................ 21
2.5 指示劑 ................................................................................................ 21
2.6 引子 .................................................................................................... 22
2.7 實驗藥品 ............................................................................................ 23
2.8 培養基成分 ........................................................................................ 24
2.9 質體 DNA 純化試劑 ( 手工法 ) .................................................... 25

2.10 染色體純化試劑 ................................................................................ 25
2.11 蛋白質膠體電泳檢體試劑 ................................................................ 25
2.12 西方墨點法材料與試劑 .................................................................... 26
2.13 穿透式電子顯微鏡試劑 .................................................................... 27
第三章、實驗方法 ........................................................................................... 28
3.1 染色體純化 ........................................................................................ 28
3.2 質體純化 ............................................................................................ 28
3.2.1 手工法 ............................................................................................ 29
3.2.2 質體萃取套組法 ............................................................................ 29
3.3 限制酶切割 ........................................................................................ 30
3.4 瓊脂膠體電泳 ( Agarose gel electrophoresis ) ................................. 30
3.5 瓊脂膠體萃取 DNA .......................................................................... 31
3.6 DNA 連接作用 ( DNA Ligation ) .................................................... 32
3.7 轉型作用 ( Transformation ) –熱休克法 ......................................... 32
3.7.1 熱休克法之勝任細胞製備 ............................................................ 32
3.7.2 熱休克法之細菌轉型作用 ............................................................ 33
3.8 轉型作用 ( Transformation ) –電穿孔法 ......................................... 33
3.8.1 電穿孔法之勝任細胞製備 ............................................................ 33
3.8.2 電穿孔法之細菌轉型作用 ............................................................ 34
3.9 細菌接合作用 .................................................................................... 34
3.10 TA-載體選殖( T-A cloning ) .............................................................. 35
3.11 蛋白質電泳分析-聚丙烯醯胺凝膠電泳 (SDS-PAGE) ................... 36
3.12 西方墨點法 ........................................................................................ 37
3.13 不同葡萄糖濃度菌株培養 ................................................................ 39
3.14 生物膜分析 ........................................................................................ 39
3.15 穿透式電子顯微鏡樣品處理 ............................................................ 40
3.15.1 固定 ............................................................................................ 40
3.15.2 負染色 ........................................................................................ 40
3.16 補回質體之建構 ................................................................................ 41
3.17 建構克雷伯氏肺炎桿菌自殺質體 .................................................... 42
3.18 同源性染色體基因重組置換 ............................................................ 43
第四章、實驗結果 ........................................................................................... 46
4.1 crp 基因剔除突變株之建構 ............................................................ 46
4.2 crp 基因剔除菌株之基因型確認 ..................................................... 47
4.3 crp 基因剔除菌株之表現型確認 ..................................................... 48
4.4 分析 crp 突變株中第三型纖毛主要結構蛋白質 MrkA 表現量 .... 48
4.5 crp 互補質體之建構 ......................................................................... 49
4.6 crp 互補株之表現型確認 ................................................................. 49
4.7 利用 TEM 觀察 crp 突變株與互補株之菌體纖毛表現 .................. 50
4.8 觀察 crp 突變株與互補株之生物膜形成 ........................................ 51
4.9 分析 crp 突變株與互補株在不同糖類濃度下第三型纖毛主要結構
蛋白 MrkA 表現量 ....................................................................................... 51
4.10 觀察 crp 突變株與互補株在不同糖類濃度下菌體纖毛的表現 .... 52
4.11 分析 crp 突變株與互補株外加 cAMP 後在不同糖類濃度下第三型
纖毛主要結構蛋白 MrkA 表現量 ............................................................... 53
4.12 分析 crp 突變株與互補株中大量表達 KPN_00353-00351 基因後在
不同糖類濃度下第三型纖毛主要結構蛋白 MrkA 表現量 ....................... 54
4.13 KPN_00353-00351 基因剔除突變株之建構 .................................... 55
4.14 KPN_00353-00351 基因剔除突變株之基因型確認 ........................ 57
4.15 分析 KPN_00353-00351 突變株第三型纖毛主要結構蛋白質 MrkA
表現量 ........................................................................................................... 57
4.16 分析臨床克雷伯氏肺炎桿菌在不同糖類濃度下第三型纖毛主要結
構蛋白質 MrkA 表現量 .............................................................................. 58
第五章、討論 ................................................................................................... 60
第六章、附圖表 ............................................................................................... 65
第七章、參考文獻 ........................................................................................... 93

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簡銘男, 陳銘仁 & 劉昌邦 糖尿病人罹患肺炎克雷伯氏桿菌肝膿瘍之病例分析.
衛生福利部疾病管制署，院內感染監視通報系統統計分析，台灣院內感染監視資
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