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標題: | 克雷伯氏肺炎桿菌K57莢膜亞型之發現與分析 Identification and characterization of K57 capsular subtypes in Klebsiella pneumoniae |
作者: | Han-Ru Yang 楊涵如 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 克雷伯氏肺炎桿菌,社區型化膿性肝膿瘍,莢膜型K57,莢膜多醣亞型,轉醯酶, Klebsiella pneumoniae,community-acquired pyogenic liver abscess (PLA),capsular serotype K57,capsular subtype,acyltransferase, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 克雷伯氏肺炎桿菌是革蘭氏陰性菌,屬於腸內菌科,可引起多種社區及院內感染,造成如:肺炎、尿道炎與菌血症等,研究指出克雷伯氏肺炎桿菌所引起的不同感染症與細菌表面的莢膜醣類型態有一定的關係。本實驗室發展的噬菌體分型法是利用可辨識特異莢膜的噬菌體及其醣解酶進行莢膜分型,其中大部分的噬菌體和其醣解酶皆可敏感的辨識特異莢膜型所有的菌株,但從標準菌株K57 (ref. K57) 所分離的噬菌體ref-K57-1和其莢膜醣類分解酵素對於標準菌株K57及臨床菌株A1142有不同的辨識情形,也因此將莢膜型K57菌株分成兩群,並推測莢膜型K57可能存在同一莢膜型下的亞型。
臨床K57菌株外圍多醣的單醣組成測定,其單醣組成比例和已發表的標準菌株推測出的組成比例並無太大差異,推測噬菌體辨識差異可能是由於莢膜醣類修飾不同造成。另外,將莢膜型K57兩群的代表菌株其完整莢膜合成區基因做比較,發現臨床菌株A1142轉醯酶 (acyltransferase) 相關的基因被transposase插入,故此基因有被破壞的可能,然而標準菌株K57具有完整的轉醯酶基因,且經由1H核磁共振 (NMR)圖譜也顯示標準菌株K57與A1142莢膜上,僅有標準菌株K57的莢膜具有O-acetylation官能基修飾。接著探討莢膜多醣的差異是否影響這兩個菌株的致病力,研究證明A1142在小鼠體內的複製能力較ref. K57強,致病力較高。 更進一步探討在A1142的莢膜合成區內將標準菌株K57的完整轉醯酶基因進行補回或置換,並觀察其O-acylation表現量、噬菌體和醣解酶的辨識情形以及致病力是否改變。成功將標準菌株K57的轉醯酶補回的A1142及A1142 LPS突變菌株,其莢膜多醣上的O-acylation表現量均有提升,且醣解酶失去對轉醯酶補回的A1142 LPS突變菌株的辨識能力;完成基因置換的A1142 LPS突變菌株,結果與基因補回的A1142 LPS突變菌株一致。選擇CPS上O-acylation修飾程度與標準菌株K57相當的突變菌株,來研究轉醯酶是否影響細菌的致病力,以基因補回的A1142 LPS突變菌株進行體內競爭性實驗,發現此突變菌株在小鼠體內的致病力與野生型A1142相較降低許多。總結目前結果,A1142與標準菌株K57於噬菌體及其醣解酶辨識情形不同,可能由於A1142 的轉醯酶基因被破壞,進而影響在莢膜多醣上的O-acylation修飾,產生莢膜型K57的莢膜亞型導致醣解酶辨識差異,且擁有完整的轉醯酶使得A1142的致病力下降,影響標準菌株K57及A1142的致病能力差異可能與轉醯酶有關。 關鍵詞:克雷伯氏肺炎桿菌、社區型化膿性肝膿瘍、莢膜型K57、莢膜多醣亞型、轉醯酶 Recently, Klebsiella pneumoniae has caused a new community-acquired emerging infectious disease, called pyogenic liver abscess, which has occurred in Taiwan and other areas. Capsular serotypes of K. pneumoniae closely associate with diseases. We established a new capsular typing method by using capsular type-specific bacteriophages and their capsule depolymerases. All of the strains determined as capsular type K57 by cps PCR genotyping could be recognized by the capsule depolymerase isolated from ref. K57, but not ref. K57. K57 strains could be divided into two groups: A and B. Therefore, we suggested that K57 capsular type might have subtypes. In this study, we focused on capsular polysaccharide composition and cps sequence of two K. pneumoniae capsular type K57 strains: ref. K57 (A group) and A1142 (B group). At first, we investigated the sugar composition of two strains by GC-MS. There were no distinct differences between ref. K57 and A1142. Then, we sequenced and aligned the cps regions of them discovering a transposase inserted to a predicted acyltansferase gene of A1142, but not that of ref. K57. After that, 1H NMR spectrum also showed ref. K57 contained O-acetylation signals, but A1142 was not. Competition index indicated A1142 was more virulent than ref. K57 significantly. Furthermore, we studied whether the robust acyltransferase in A1142 capsular polysaccharide synthesis lead to changes of O-acylation level, bacteriophage/capsule depolymerase recognition and virulence. We discovered the capsule depolymerase lost the ability to recognize A1142 LPS mutant with acyltransferase complementation and replacement. After that, using the A1142 LPS mutant with acyltransferase complementation whose O-acylation level of CPS was almost equal to that of ref. K57 investigated the change of virulence by in vivo competition index. This result proved robust acyltransferase from ref. K57 in A1142 would decrease the bacterial load in BALB/c extremely. In conclusion, ref. K57 and clinical isolate A1142 have different O-acylation modification of CPS and acyltransferase in cps region. A1142 might be a subtype of capsular type K57 in K. pneumoniae. Acyltransferase was probably one of the reasons leading the different pathogenicity between ref. K57 and A1142. Keywords: Klebsiella pneumoniae, community-acquired pyogenic liver abscess (PLA), capsular serotype K57, capsular subtype, acyltransferase |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61524 |
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