降解克雷伯氏肺炎菌K2(A4528)莢膜多醣之嗜菌體酵素的純化及其生化特性的探討

Purification and biochemical characterization of a bacteriophage hydrolase to degrade capsular polysaccharide of PLA Klebsiella pneumoniae K2 (A4528)

張愉珮(Yu-Pei Chang)

指導教授 ： 吳世雄

國立臺灣大學/理學院/化學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10696

摘要

過去二十幾年來，在亞洲地區特別在台灣，導致化膿性肝膿瘍的克雷伯氏肺炎菌是社區型及院內感染常見的病原體，有相當高的發病率以及致死率。臨床研究顯示，莢膜血清型K1及K2就約佔77.6 %。隨著會分泌廣效性乙內醯胺酶的克雷伯氏肺炎菌比率不斷增加，多重抗藥性的問題愈趨嚴重，使得治療更加地棘手，由細菌莢膜多醣製成的醣複合疫苗可能是一個解決現在困境的策略。然而多醣因為分子量太大，不易架接至載體蛋白成為有效抗原，所以我們利用嗜菌體的尾巴蛋白將克雷伯氏肺炎菌A4528(莢膜血清型K2)的莢膜多醣水解成寡糖，以提升架接產率，成為有效抗原。
在此論文中，主要探討K2水解酶的生物物理特性及其與受質K2莢膜多醣的相互作用。藉由嗜菌體篩選，兩個K2莢膜多醣水解酶被選殖出來，其兩者序列的相似度只有25.4 %，並探討兩者間特性的比較: 首先進行圓二色性光譜以及分析超速離心來偵測其二級結構和蛋白質自然狀態，此兩個水解酶的二級結構皆為β折疊以及以三聚物形式存在；另外也使用X光晶體繞射學來研究晶體結構，目前收到一組K2-1水解酶的結晶資訊，其解析度為3.58 A。而水解酶切割莢膜多醣的活性可藉由3,5-二硝基水楊酸來定量水解後新增還原端的含量，K2-1的最佳緩衝液為20 mM NaOAc/MES/HEPES、100 mM NaCl，pH值6，在溫度為37 ℃下有最佳的酵素活性；至於K2-2則是在緩衝液為20 mM sodium citrate、100 mM NaCl，pH值5，在溫度為50 ℃下有最大的活性。並利用質譜儀來鑑定經由水解酶反應後多醣片段的型式，從質譜圖推斷無論是K2-1或K2-2水解片段, 主要以單聚體(四寡醣)為主，亦有二聚體(八寡醣)。最後，利用核磁共振來解析K2-2水解酶的切點，從各類不同圖譜推斷其切點位於葡萄糖和甘露糖中間的1,4-β-1,4連結。

關鍵字

克雷伯氏肺炎菌 ； 化膿性肝膿瘍 ； 醣複合疫苗 ； 莢膜血清型K2 ； 莢膜多醣 ； K2水解酶

並列摘要

Klebsiella pneumoniae is a common cause of community-acquired and nosocomail infections that causes pyogenic liver abscess (PLA) in Asian over two decades, especial in Taiwan area, and leads to significant morbidity and mortality. The clinic investigation showed that K. pneumoniae serotypes K1/K2 are predominant among capsular serotypes of virulent PLA strains, around 77.6%. Besides, infection caused by extended-spectrum β-lactamase (ESBL)-producing pathogens, particularly K. pneumoniae, are increasing. Because of resistance to numerous antimicrobial agents, treatment turns into a critical medical issue. Hence, glycoconjugate vaccines which source from capsular polysaccharide of bacteria would offer one kind of solution to this bacterial infection. Since polysaccharide is too large to be conjugated to a carrier protein, hard to become an effective antigen, the depolymerization of capsular polysaccharide would result in the increasing yield of the coupling. Thus, we utilized a tail fiber enzyme from bacteriophage to cleave the capsular polysaccharide of K. pneumoniae A4528 (serotype K2) into smaller sugar fragments, in order to increase the effective conjugation during antigen preparation.
In this study, two low-identical K2 hydrolases (25.4 %) were cloned from different K2-bacteriophages. Based on biophysical instrumentation, including CD and AUC, the secondary structures and native state of the proteins were determined. The conformation of K2 hydrolases are primarily in β-sheet and exist as trimers. Besides, X-ray crystallography was applied to dissect the atomic structure of K2 hydrolase. To date, the crystallographic data of K2-1 hydrolase was collected which resolution was 3.58 A. Otherwise, in the aspect of interaction between enzyme and substrate, the enzymatic activity could be measured by 3,5-dinitrosalicylic acid which enable to quantify the forming of new reducing end of sugars once hydrolase digested CPS. With a series of screening, the optimal conditions of the two hydrolases were determined. K2-1 and K2-2 have the maximum activities in optimal conditions: 20 mM NaOAc/MES/HEPES buffer with 100 mM NaCl at pH 6 and 37 ℃, and 20 mM sodium citrate buffer with 100 mM NaCl at pH 5 and 50 ℃, respectively. To understand the fragments of hydrolase-digested CPS, mass spectrometry was applied. From the results of MALDI-TOF spectra, the major products of hydrolase-digested CPS are one-repeat unit (monomer; tetrasaccharides) and two-repeated units (dimer; octasaccharides) as minor. Additionally, the cutting site of hydrolase-digested CPS was identified by NMR analysis. After resolving NMR spectra, we found that the cutting site of K2-2-digested CPS is located in the β-1,4 linkage between glucose and mannose.

並列關鍵字

Klebsiella pneumoniae ； pyogenic liver abscess ； glycoconjugate vaccines ； K2 serotype ； capsular polysaccharide ； K2 hydrolase

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