Purification and biochemical characterization of a bacteriophage hydrolase to degrade capsular polysaccharide of PLA Klebsiella pneumoniae K2 (A4528)
Date Issued
2014
Date
2014
Author(s)
Chang, Yu-Pei
Abstract
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.
Subjects
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
SDGs
Type
thesis
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