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研究生(外文):Yu-Pei Chang
論文名稱(外文):Purification and biochemical characterization of a bacteriophage hydrolase to degrade capsular polysaccharideof PLA Klebsiella pneumoniae K2 (A4528)
指導教授(外文):Shih-Hsiung Wu
口試委員(外文):Po-Huang LiangKuo-Feng HuaChun-Hua Hsu
外文關鍵詞:Klebsiella pneumoniaepyogenic liver abscessglycoconjugate vaccinesK2 serotypecapsular polysaccharideK2 hydrolase
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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&;#37238;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&;#37238;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&;#37238;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&;#37238;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&;#37238;的結晶資訊,其解析度為3.58 A。而水解&;#37238;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&;#37238;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&;#37238;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。

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.

謝誌 I
摘要 II
Abstract III
List of Figures VII
List of Tables IX
Abbreviation Table X
1 Introduction 1
1-1 Klebsiella pneumoniae 1
1-2 Pyogenic liver abscess (PLA) 3
1-3 Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae 3
1-4 Capsular polysaccharide (CPS) 4
1-5 Glycoside hydrolase 6
1-6 Glycoconjugate vaccine 8
1-7 Purpose of this research 9
2 Materials and Methods 11
2-1 Protein expression and purification 11
2-2 Phylogenetic analysis and sequence alignment of K2 hydrolase 12
2-3 Circular dichroism (CD) spectra 12
2-4 Analytical ultracentrifugation (AUC) analysis 13
2-5 X-ray crystallography 14
2-6 Capsular polysaccharide purification 15
2-7 Quantification of reducing ends with 3,5-dinitrosalicylic acid (DNSA) assay 15
2-8 MALDI-TOF spectra 17
2-9 MS/MS spectra 18
2-10 NMR spectroscopy 18
3 Results 20
3-1 The sequence relationship of two K2 hydrolases of PLA K. p and other related hydrolases 20
3-1-1 Phylogenetic analysis of K2 hydrolases 20
3-1-2 Protein sequence alignment of K2-1 and K2-2 22
3-2 Biophysical characterization of K2-1 and K2-2 23
3-2-2 Analytical ultracentrifugation analysis 24
3-3 X-ray crystallography for K2 hydrolases 27
3-4 Biochemical assay for K2 hydrolase 29
3-4-1 Optimal pH screening 29
3-4-3 Optimal buffer screening 33
3-4-4 Comparison of the activity between K2-1 and K2-2 35
3-5 MS and MS/MS analysis of hydrolase-digested capsular polysaccharide 36
3-6 NMR analysis of K2-2-digested CPS 39
4 Conclusion and Discussion 48
5 References 56

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