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標題: | 克雷伯氏肺炎桿菌中三套rmpA/A2基因之功能分析 Functional characterization of three rmpA/A2 loci in Klebsiella pneumoniae |
作者: | You-Ci Chen 陳又綺 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 克雷伯氏肺炎桿菌,rmpA/A2基因,莢膜,cps gene cluster, Klebsiella pneumoniae,rmpA/A2,capsule,,cps gene cluster, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 近二十年來,在台灣出現了一種新型的侵襲性克雷伯氏肺炎桿菌,經由社區感染,會引起典型的臨床表現—原發性肝膿瘍(community-acquired pyogenic liver abscess,PLA)合併菌血症,莢膜為菌株之主要致病因子之一。rmpA/A2基因能促進菌株的莢膜表現量進而影響黏性型態與致病力,在從台大醫院收集的引發原發性肝膿瘍菌株中rmpA/A2之比例為100%(60/60),而在非組織侵襲性菌株中為22%(7/32)。在本實驗室代表性菌株NTUH-K2044中有三套rmpA/A2基因,分析三套rmpA/A2基因在臨床菌株中之分佈,發現以質體pK2044上的rmpA及rmpA2基因分布比例較高。以NTUH-K2044建構染色體上rmpA基因剔除株(Δ chromosome rmpA)、質體pK2044上rmpA基因剔除株(Δ plasmid rmpA)與質體pK2044上截斷形式的rmpA2基因剔除株(Δ plasmid rmpA2),結果顯示Δ chromosome rmpA突變株對黏性、莢膜生合成量及其基因組表現無影響;Δ plasmid rmpA突變株能明顯降低黏性型態及莢膜生合成基因組表現;Δ plasmid rmpA2突變株之莢膜生合成基因組表現有些微上升,但不影響黏性型態及莢膜生合成量。而在以腹膜腔注射及胃內感染方式探討對小鼠致病力的結果中,Δ plasmid rmpA突變株與野生株亦無明顯差異,然而在小鼠體內競爭實驗中,則發現Δ plasmid rmpA突變株在肝臟及脾臟的散佈生長情形明顯較野生株強。另外,從三株基因剔除株中分析三套rmpA/A2基因的表現量結果得知,只有在失去質體 pK2044 rmpA基因時,會降低染色體rmpA及質體pK2044 rmpA2的表現量;染色體rmpA基因對質體 pK2044 rmpA及rmpA2基因的表現量影響不大;質體 pK2044 上截斷的rmpA2則有些微抑制另兩套rmpA基因的表現量。染色體rmpA基因與質體 pK2044 rmpA基因序列相同度為92%,但只有質體 pK2044 rmpA基因會影響莢膜生合成表現,將野生株中的質體 pK2044 rmpA置換為染色體rmpA後,莢膜基因組表現量有明顯下降。因此,由本實驗結果可知,在NTUH-K2044菌株內三套rmpA/A2中,只有破壞質體 pK2044rmpA會降低莢膜表現量及黏性,並且增強菌株於小鼠體內器官中散佈生長的情形;而造成染色體rmpA及質體 pK2044 rmpA兩者在調控黏性功能上的不同源於兩者之基因序列不同。 There are three copies of rmpA/A2 (regulator of the mucoid phenotype A) genes located in the chromosome or large plasmid of Klebsiella pneumoniae NTUH-K2044 strain. This gene has been reported to be involved in the biosynthesis of capsular polysaccharide. A copy of rmpA is located on the chromosome. A rmpA and a truncated rmpA2 are located at the pK2044 plasmid of NTUH-K2044. In order to study the specific biological function of these three rmpA/A2 genes, we generated three rmpA/A2 single deletion mutants. We found that the deletion of plasmid rmpA resulted in decreases of K1 CPS-related gene expression and colony mucoviscosity. Comparing with the wild-type strain, the chromosome rmpA deletion mutant showed no significant difference in K1 CPS-related gene expression and colony mucoviscosity, whereas the plasmid rmpA2 mutant had an increase in the K1 CPS-related gene expression but not in the K1 CPS synthesis. In in vivo virulence analysis, we used intraperitoneal inoculation infection in BALB/c mice model to perform in vivo competition assay. The results revealed that colonization ability of plasmid rmpA mutant was stronger than that of wild type in liver, and spleen. This implied that the decrease of the K1-CPS synthesis of K. pneumoniae in Δplasmid rmpA mutant might contribute to facilitate in vivo colonization of K. pneumoniae. In addition, chromosome rmpA and plasmid rmpA2 mRNA expression was decreased in the plasmid rmpA2 mutant compared than the wild-type strain. Chromosome rmpA gene and plasmid rmpA gene shared a 92% identity on DNA sequence. However, only plasmid rmpA was capable of up-regulating the expression of cps gene. While plasmid rmpA was replaced by chromosome rmpA in NTUH-K2044, the expression of cps genes decreased. We concluded that plasmid rmpA had the ability to enhance CPS synthesis, but chromosome rmpA and plasmid rmpA2 had no influence on CPS synthesis. Moreover, destruction of plasmid rmpA promoted bacterial colonization in liver and spleen.The distinct ability of regulating cps region between chromosome rmpA and plasmid rmpA is the result of their different sequences. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45379 |
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