Purification and biochemical characterization of a bacteriophage lyase specific to cleave the capsular polysaccharides of PLA Klebsiella pneumoniae NTUH-K2044

在過去二十年中，克雷伯氏肺炎菌(Klebsiella pneumonia)在亞洲族群中是造成肝膿瘍的主要微生物之一。由於這類型的肝膿瘍通常會併發敗血症、菌血症、轉移性腦膜炎或眼內炎，所以其造成的死亡率也是很顯著的。臨床的研究結果顯示K1以及K2這兩種莢膜血清型的菌株是導致肝膿瘍的主要菌株,大約佔了50-85%。本研究的主要目標是去建構一種以莢膜多醣和蛋白接合在一起的疫苗，期望能有效預防經由克雷伯氏肺炎菌所導致的肝膿瘍。一般來說，由於莢膜多醣是一聚合物，其分子量和長度過於龐大，所以無法有效率地與攜帶蛋白(carrier protein)接合，不過藉由去聚合化作用將可以提昇接合的效率。在我們之前的研究中顯示，藉由化學的方法進行切割將會導致莢膜多醣上的修飾基例如：乙醯化、丙酮酸化的分離，這將使莢膜多醣失去其引發免疫反應的功用。 在我們現在的研究中，我們將建構好的噬菌體K1裂解酶(K1 lyase)表現以及純化出來。其相對活性可以藉由波長232nm的改變來測量。其最適pH值以及溫度分別是7.5以及30℃。鎂離子對於K1裂解酶的活性有實質上的幫助，不過其角色也可以被鈷離子或鎳離子所取代。我們也建構了可以更穩定且有效率的對莢膜多醣做切割的K1裂解酶突變株。藉由質譜的分析我們可以證實藉由K1裂解酶的作用我們可以對莢膜多醣進行切割卻不會使修飾基與之分離。同時我們也使用毛細管電泳(capillary electrophoresis)動態地偵測被切割後的莢膜多醣片段。Klebsiella pneumoniae is one of the microorganisms causing pyogenic liver abscess (PLA) in Asian populations over two decades. This kind of PLA is associated with significant mortality because of developing into sepsis, bacteremia, metastatic meningitis or endophthalmitis. The clinic investigation showed that K. pneumonia serotypes K1 and K2 are predominant among capsular serotypes of PLA strains, around 50-85％. The major purpose of this study is to construct a capsular polysaccharide (CPS)-protein conjugate vaccine for prevention of K. pneumonia PLA. Since CPS is too large to be conjugated to carrier proteins, the depolymerization of CPS will increase the coupling efficiency to the carrier protein. According to our previous study, the chemical cleavage will destruct the CPS modifications including acetylation and pyruvation and, meanwhile, abolish the immune responses. In the present study, the K1 lyase from bacteriophage was cloned, expressed and purified by the traditional methods. The relative K1 lyase activity can be measured by UV absorbance at 232 nm. The optimal pH and temperature of K1 lyase is 7.5 and 30℃, respectively. The magnesium ion is essential for the activity of K1 lyase, but, to some extent, can be replaced by other metal ions such as cobalt and nickel. We also constructed a mutant of K1 lyase which could cleave the CPS more stably and efficiently. Based on the analysis of MS, acetylation and pyruvation in CPS remain intact after enzymatic cleavage. Capillary electrophoresis was also used to kinetically detect the fragmentation of CPS