臺灣博碩士論文加值系統

克雷伯氏肺炎桿菌NTUH-K2044引起之肝膿瘍併發轉移性病灶是種台灣近年來新興的感染性疾病，這種人類細菌性病原外層包覆著多醣類物質成為物理性屏障，這種醣鞘結構稱為莢膜多醣體(CPS)，是一種重要的致病因子，用來躲避人體免疫系統的攻擊。文獻指出，與莢膜多醣體合成有關的基因組位於cps (capsule polysaccharide synthesis) 基因座上，其中包含wzb基因，研究發現，剔除wzb基因後的菌株會失去莢膜的包覆，因而推測wzb基因參與莢膜多醣體合成過程的調控，此外，根據蛋白質序列比對分析，Wzb蛋白也被認為是一種低分子量之酪胺酸去磷酸脢(LMW-PTP：Low molecular weight protein tyrosine phosphatase)。
為了釐清Wzb蛋白利用蛋白質酪胺酸磷酸化作用調控莢膜多醣體生合成時所扮演的角色，首先，將造成肝膿瘍之克雷伯氏桿菌NTUH-K2044品系的wzb基因選殖至適當基因載體，並在其C端同時加上His以及HA胜肽標記，此外，依據其催化區設計了Wzb蛋白的受質捕捉(substrate-trapping)突變株，分別為WzbC9S、WzbD115A及WzbC9S/D115A。Wzb 蛋白的去磷酸化活性在pH 5.5環境下達到最高，於26.8°C所測得的動力學常數：Km、Vmax、Kca以及Kcat/Km 分別為1.35 mM、 34.8 μmole min-1 mg-1、641.1 min-1以及475 mM-1 min-1。在酵素活性抑制實驗中發現，Wzb不會受到高濃度之NaF與EDTA的抑制(10 mM)，相反地，在10 μM H2O2、1 μM vanadate、1 mM Iodoacetamide的處理下，Wzb的活性則受到完全抑制。從以上這些結果證實克雷伯氏桿菌NTUH-K2044的Wzb蛋白可被歸類為低分子量酪胺酸去磷酸脢。
胞外去磷酸化實驗結果指出，許多內生性酪胺酸磷酸化蛋白質會受到Wzb的去磷酸化作用，暗指這些未知蛋白都有可能是Wzb蛋白的目標物且受其調控，藉由未知蛋白的身分鑑定來了解細菌體內受到酪胺酸磷酸化系統調控的細胞活性，為了揭露Wzb蛋白的天然受質(natural substrates)的真面目，低分子量酪胺酸去磷酸脢首次被用在受質捕捉實驗裡，結果不如預期，未能成功鑑定出任何酪胺酸磷酸化蛋白，然而，透過直接測試的方法得知，具有自體酪胺酸磷酸化激脢活性的Wzc是屬於Wzb的一個受質，然而，仍然有許多天然受質鑑定上的障礙需要被克服，為了更進一步了解酪胺酸磷酸化系統與細菌莢膜多醣體生合成的關聯性抑或是其他相關的細胞反應，未來我們必須找到一些更適合的研究方法藉此回答這些問題，受質鑑定的研究仍然持續地在進行中。

Liver abscess with metastatic complications caused by Klebsiella pneumoniae (K. pneumoniae) is an emerging infectious disease in Taiwan in the recent years. The human bacterial pathogen was wrapped in a physical barrier of exopolysaccharides. The sugar coated structure, termed capsular polysaccharides (CPS), is an important virulence factor which protects the pathogen from attack by the host immune system. It has been documented that a cassette of genes involved in CPS development are gathered at the capsule biosynthesis locus (cps). The Wzb gene is localized within such gene cluster which determines synthesis and assembly of CPS. It has been suggested that wzb is involved in the regulation of CPS biosynthesis, since the capsule is eliminated in the wzb knockout strain. Furthermore, Wzb is also considered as a kind of low molecular weight protein tyrosine phosphatases (LMW-PTP) based on the protein sequence alignment.
In order to confirm the role of Wzb in regulating bacterial CPS biosynthesis through protein tyrosine phosphorylation, we first cloned the Wzb from the liver abscess strain K. pneumoniae NTUH-K2044 and tagged the protein with both histidines (His) and haemagglutinin epitop (HA) at the C-terminus. Meanwhile, substrate-trapping mutants, C9S, D115A and C9S/D115A, were also generated based on the information of the active site. The maximal enzymatic activity of Wzb was achieved at a pH of 5.5 and the corresponding kinetic constants Km, Vmax, Kcat and Kcat/Km, measured at 26.8°C, were 1.35 mM, 34.8 μmole min-1 mg-1, 641.1 min-1and 475 mM-1 min-1, respectively. The inhibition assay revealed that Wzb is not inhibited by NaF and EDTA, even at a high concentration (10 mM). On the contrary, pre-incubation with 10 mM H2O2 or 1 μM vanadate or 1 mM indoacetamide caused the phosphatase to lose its activity completely. These results confirmed that Wzb of K. pneumoniae NTUH-K2044 can be classified as a LMW-PTP.
In vitro dephosphorylation assay indicated that several endogenous tyrosine phosphorylated proteins can be dephosphorylated by Wzb, suggesting that these unknown proteins are potential targets of Wzb and governed by it. On the other word, the cellular activity regulated by tyrosine phosphorylation system in bacteria can be clarified by substrate identification. In order to uncover the endogenous substrates of Wzb, we perform substrate-trapping experiments to pull them out where the LMW-PTP is first adopted. Against all expectations, substrate-trapping analysis falls to identify any endogenous tyrosine phosphorylated proteins, however the tyrosine-autokinase, Wzc, can serve as a substrate of Wzb that was verified by a straightforward manner. There are still several obstacles to overcome in the recognition of natural substrates. For further understanding of the precise roles of tyrosine phosphorylation system in bacterial CPS biosynthesis or other associated cellular responses, more precise experimental methods must be exploited in the future to answer those questions. Our investigation on the substrate identification is still ongoing.

中文摘要 I
ABSTRACT III
INTRODUCTION 1
1. KLEBSIELLA PNEUMONIAE 1
2. BACTERIAL EXOPOLYSACCHARIDES 3
3. PHOSPHORYLATION IN BACTERIA 6
4. CHARACTERIZATION OF PTPS SUBSTRATES 9
MATERIAL AND METHOD 12
1. BACTERIAL STRAINS AND PLASMID 12
2. CULTURE MEDIUM AND GROWTH CONDITION 12
3. CLONING OF THE PUTATIVE LMW-PTP WZB 12
4. SITE-DIRECTED MUTAGENESIS 13
5. OVEREXPRESSION AND PURIFICATION OF WZB 14
6. ASSAY FOR PROTEIN TYROSINE PHOSPHATASE ACTIVITY 15
8. INHIBITION ASSAY 17
9. IMMUNOBLOTTING ANALYSIS 17
10. IMMUNOPRECIPITATION 18
11. PROTEIN IDENTIFICATION 19
RESULT 20
1. PURIFICATION OF THE PUTATIVE LMW-PTP WZB FROM K. PNEUMONIAE 20
1.1 Cloning of the wzb gene 20
1.2 Multiple sequence alignment of LMW-PTPs from various organisms suggests that Wzb is a LMW-PTP 21
1.3 Generation of the Wzb mutants 21
2. THE ENZYMATIC ACTIVITY OF WZB IS INFLUENCED BY VARIOUS FACTORS 22
2.1 The pH effect on Wzb activity reveals that the maximal enzymatic activity was achieved at pH 5.5 22
2.2 Kinetics of Wzb was performed that the corresponding kinetic constants were Km: 1.35 mM, Vmax: 34.8 μmole min-1 mg-1, Kcat: 641.1 min-1 and Kcat/Km: 475 mM-1 min-1 22
2.3 Wzb was a tyrosione phosphatase determined by enzyme inhibition assay 23
3. ENDOGENOUS SUBSTRATES FOR WZB 24
3.1 There was no difference in growth rate among the K. pneumoniae NTUH-K2044 wildtype, magA- and wzb- mutant strains 24
3.2 Differential profiles of protein tyrosine phosphorylation were existed between wildtype and magA- mutant strains 25
3.3 Wzb potentially dephosphorylate endogenous proteins phosphorylated on tyrosine residues 26
3.4 Substrate-trapping analysis failed to capture the natural substrates that can be dephosphorylated by Wzb 26
3.5 It is a popular occurrence in several bacterial species that Wzc is a substrate of Wzb 27
DISCUSSION 30
FIGURE CONTENTS 37
REFERENCES 56

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