由造成肝膿瘍之克雷伯氏肺炎桿菌分離一高抗原性分泌蛋白質

克雷伯氏肺炎桿菌是院內感染常見的致病菌。近二十年來，臺灣出現一種新型的侵襲性克雷伯氏肺炎桿菌原發性肝膿瘍感染，並有逐年增加的趨勢。我們以感染克雷伯氏肺炎桿菌而造成肝膿瘍的恢復期病人血清，偵測由細菌培養液收取而來的分泌蛋白質，發現一個具高抗原性的標的蛋白質。隨後以蛋白質體學的方法，先用二維電泳將此蛋白質分離，再用N端蛋白質定序、基質輔助雷射脫附游離/ 飛行時間質譜分析和液相層析質譜質譜分析等方法鑑定。最後，在液相層析質譜質譜分析的結果，得到標的蛋白質的部分胜肽序列，在和2003年年底剛完成的克雷伯氏肺炎桿菌臺大菌株NTUH-K2044完整基因體序列作比對之後，終於鑑定出此標的蛋白質的基因位於克雷伯氏肺炎桿菌大質體上第307個開放讀架（即KPP307）。KPP307的基因大小為921個鹼基對，轉譯成306個胺基酸、分子量大小為32 kDa的未知蛋白質。生物資訊學的分析結果顯示KPP307在N端有一段含25個胺基酸的訊息胜肽。我們經由蛋白質表現和西方墨點法重新確認標的蛋白質。另一方面，我們發現相較於健康人，在目前所有感染克雷伯氏肺炎桿菌而造成肝膿瘍的恢復期病人血清樣本中，利用西方墨點法皆可檢測到1：1000以上高效價的抗KPP307抗體，顯示此蛋白質在診斷侵襲性克雷伯氏菌感染上的潛在價值。然而，KPP307在侵襲性克雷伯氏肺炎桿菌致病機轉上所扮演的角色為何則有待進一步研究。

關鍵字

肝膿瘍；高抗原性；克雷伯氏肺炎桿菌；分泌蛋白質

並列摘要

Klebsiella pneumoniae is a common pathogen, causing nosocomial infections. Over the past two decades, a new type of invasive K. pneumoniae disease has emerged in Taiwan that typically presents a community-acquired primary liver abscess. In this study we attempted to analyze immunodominant proteins secreted by K. pneumoniae strains causing liver abscess. We found a target protein from culture supernatants by immunoblot analyses with convalescent-phase patient sera. Subsequently we identified the protein by proteomic approach including 2DE, N-terminal protein sequencing, MALDI-TOF MS and LC-MS/ MS. LC-MS/ MS identified the target protein by comparing the partial peptide sequence with the full genome sequence of clinical strain NTUH-K2044. A novel 921-bp locus, KPP307, which encoded a 32-kDa unknown protein, was the 307th open reading frame located on the large plasmid of K. pneumoniae. Bioinformatic analyses revealed that KPP307 started with a signal peptide composed of 25 amino acids. All convalescent-phase patient serum samples collected so far contained Western-blot antibodies at a titer of 1:1000 or greater, indicating potential applications for this protein in diagnosis of invasive K. pneumoniae infections. However, the role of KPP307 in virulence mechanism awaits further studies.

並列關鍵字

Klebsiella pneumoniae ； liver abscess ； immunodominant ； secreted protein

參考文獻

38. Williams, P., Ketley, J. & Salmond, G. Bacterial Pathogenesis. Academic Press. California. 2nd printing. pp620 (1998)

2. Albano, F. et al. Structural and functional features of modified heat-stable toxins produced by enteropathogenic Klebsiella cells. Pediatr. Res. 48, 685-690 (2000)

3. Bumann, D. et al. Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori. Infect. Immun. 70, 3396-3403 (2002)

4. Chen, Y. T. & Peng, H. L. Complete DNA Sequence and Annotation of the Large Virulence Plasmid pLVPK of Klebsiella pneumoniae CG43. [unpublished]

5. Chou, H. C. et al. Isolation of a chromosomal region associated with allantoin metabolism and liver infection. Infect. Immun. 72, 3783-3792 (2004)

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由造成肝膿瘍之克雷伯氏肺炎桿菌分離一高抗原性分泌蛋白質

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