克雷伯氏肺炎菌NTUH-K2044及其莢膜多醣感染之HepG2細胞磷酸化蛋白質體之研究

Klebsiella pneumonia （K. pneumoniae）克雷伯氏肺炎菌，為一常見的腸內菌科，屬於革蘭式陰性菌，經常引起醫院內群聚感染。近幾十年來，K. pneumoniae在臺灣地區造成肝膿瘍（PLA）病例增加，甚至細菌轉移至腦產生腦膜炎或眼炎。K. pneumoniae最大之特徵莫過於細菌細胞壁外層的莢膜多醣，這一層多醣構造使得細菌具有高度黏性，也是重要之毒性因子。若K. pneumoniae一旦為高黏性之菌株，就極有可能具有抵抗血清中免疫細胞之攻擊，對於宿主就會產生嚴重之影響。而K. pneumoniae造成肝膿瘍之機制又是如何，是本篇論文所之目標。本實驗使用從肝膿瘍患者上分離到之菌株K. pneumonia NTUH-K2044，及一肝癌細胞株HepG2進行研究。研究發現，HepG2一旦被K. pneumonia NTUH-K2044感染，細胞內酪氨酸（tyrosine）磷酸化蛋白質表現量有增加的情形，而若是由pneumonia NTUH-K2044之莢膜多醣感染HepG2細胞，細胞內酪氨酸磷酸化蛋白質表現量則在2至2.5小時左右達到最高峰。而HepG2經K. pneumonia NTUH-K2044感染6小時會發現細胞數量逐漸減少、死亡，藉由流式細胞儀觀察細胞DNA，不論感染時間多寡，均維持完整之狀態，因此在此判斷細胞可能活化另外之蛋白質路徑而使細胞邁向死亡，而不是使DNA斷裂。細胞可藉由蛋白質的磷酸化與去磷酸化轉換蛋白質的功能或活性，以達成調控細胞內訊息傳遞及生理活動，肝膿瘍的產生是否與這些路徑有關，是可以探討的方向。本研究最後希望藉由磷酸化蛋白質體之方式研究HepG2細胞在有無K. pneumonia NTUH-K2044 CPS刺激下觀察蛋白質磷酸化是否有差異，及找出相關之訊息傳遞路徑。實驗結果顯示膠內挖取得到之蛋白質並非西方墨點法所壓到之蛋白質，而以serine及threonine磷酸化之蛋白質居多，而無發現符合之酪氨酸磷酸化蛋白質。

關鍵字

克雷伯氏肺炎菌；莢膜多醣；肝膿瘍；磷酸化蛋白質體學

並列摘要

Klebsiella pneumoniae, an enterobacterium, is a gram-negative strain of bacteria that belongs to enterobacteriae. K. pneumoniae is responsible for most cases of hospital-acquired pneumonia, The bacteriae often causes severe pyogenic liver abscess （PLA） that is accompanied with metastatic meningitis or endophthalmitis recently. However, it remains obscure how K. pneumoniae may become virulent in human host and result in liver abscess. It is tentative to suggest that its toxicity rises from its remarkable mucoviscosity, which is modulated by the level of external capsule polysaccharides (CPS). It is possible that once K. pneumoniae attains hypermuscosity, it could not be targeted by immune cells, and thereby leads to severe consequences. In our study, we intend to find the missing link between the CPS of K. pneumonia and PLA pathogenesis by investigating the level and extent of protein phosphorylation in the infected HepG2 cells. We use a virulent strain K. pneumonia NTUH-K2044 which was isolated from a PLA patient in Taiwan, and a hepatoma cell line HepG2 as experiment model. When HepG2 was infected by K. pneumonia NTUH-K2044, the expression of phosphotyrosine protein was immediately upregulated. While HepG2 was infected by CPS extracted from K. pneumonia NTUH-K2044, phosphotyrosine proteins increased to optimum in 150 min. After K. pneumonia NTUH-K2044 infection for 6 hours, HepG2 died. We used a flow cytometry to identify whether cells died of apoptosis. The result showed that the DNA of cells was intact, we speculated, therefore, that cells apoptosis were activated via other pathways. Phosphorylation of proteins plays an important role in cellular process by modulating protein function and transmitting signals within cellular pathways and networks. To identify the phosphoproteins involve in the CPS infection, we use MS/MS to study the phosphoproteome of infected HepG2. Because the signal intensity phosphoserine and phosphothreonine protein was too high and put a cover over phosphotyrosin in MS/MS analysis, there was no remarkable proteins found in in-gel digestion.

並列關鍵字

Klebsiella pneumoniae ； capsule polysaccharide ； liver abscess ； phosphoproteome

參考文獻

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克雷伯氏肺炎菌NTUH-K2044及其莢膜多醣感染之HepG2細胞磷酸化蛋白質體之研究

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