Helicobacter pylori is a Gram-negative microaerophilic bacterium which infects more than 50% world population and is recognized as an important etiological pathogen of human stomach diseases including chronic inflammation of stomach and duodenum, chronic gastritis, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Although the combinational antibiotic therapy is frequently applied as the remedy for H. pylori infection, it has been shown that the prevalence of antibiotic resistance among H. pylori isolates is increasing. Therefore, the development of new antibiotics against this notorious bacterium seems important.
We considered the enzymes in coenzyme A (CoA) biosynthetic pathway as the potential targets for development of novel antibiotic drugs. CoA, an important intracellular carrier of activated acyl groups in various metabolic pathways, is synthesized from pantothenate (vitamin B5) in five enzymatic steps. Previous researches indicated that phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3) is the regulatory point of the CoA biosynthetic pathway and there is no significant sequence homology between bacterial and mammalian orthologs. Accordingly, PPAT is really a potential antibiotic drug target.
In this study, we identified the H. pylori open reading frame (ORF) hp1475 codes for the PPAT, which has been considered coding an enzyme associated with lipopolysaccharide biosynthesis in previous researches. We cloned the gene and overexpressed in Eschericha coli. The recombinant enzyme was purified to homogeneity with the subunit molecular weight approximating 19.7 kDa. The gel filtration studies suggested that HP1475 protein has a native molecular mass of 88.39 ± 2.44 kDa (4.49 ± 0.12 folds of subunit molecular weight), indicating that the native HP1475 protein is likely a tetramer. The experimental result of analytical ultracentrifugation indicated that H1475 has a native molecular mass of 79.86 ± 3.37 kDa (4.053 ± 0.17 folds of subunit molecular weight), under the present experimental conditions.
The PPAT activity was characterized through the reverse enzymatic activity assay and nuclear magnetic resonance spectroscopy (NMR). The results confirmed that the purified protein could catalyze the reverse reaction and convert dephospho-CoA (dPCoA) and pyrophosphate to 4’-posphopantetheine and ATP. In addition, the secondary structure of protein was analyzed with circular dichroism spectroscopy (CD) and the data indicated that HP1475 contains 63.01 ± 10.04 % α-helix and 3.87 ± 2.40% β-strand.
The fractionation result from point mutation studies on highly conserved residues among species in the heterogeneous system suggested that Tyr7 and Arg88 are important for protein solubility. Analyses of HP 1475 point-mutated proteins in a discontinuous native protein gel electrophoresis system suggested that Arg88 is important for subunit assembly. The His18 is related to protein stability and secondary structure as indicated by the result of native protein gel electrophoresis assay and CD experiment respectively. The effects of point-mutated residues on the reverse catalytic activity of the protein implied that these conserved amino acid residues are important for the function of protein. These residues are located in the activity pocket predicted by pymol software and are related to the substrate biding ability but have no influence on the protein secondary structure. Finally, an antisense RNA interference system was used to study the function of HP1475 in vivo. The results showed that expression of antisense hp1475 RNA achieved 50~60% knockdown of HP1475 protein levels in the E. coli BL21 (DE3) expression system. When this knockdown system was directly applied in H. pylori, a similar knockdown result was also observed. Furthermore, the generated HP1475 knockdown H. pylori strain was found to decrease growth efficiency and have a highly level of CoA compared with wild type H. pylori 26695.

胃幽門桿菌是一種螺旋狀的革蘭氏陰性微好氧菌。它被認定為重要的人類胃部致病菌，世界上有超過百分之五十的人口曾被此菌感染。胃幽門桿菌感染可能造成慢性胃、十二指腸發炎或潰瘍的情形，甚至造成胃腺癌以及胃黏膜組織淋巴瘤。複合性的抗生素治療可以達到治癒胃幽門桿菌感染的目的，但是先前研究指出具有抗藥性的胃幽門桿菌菌株大幅增加，因此對於此惡名昭彰的致病菌，開發新的抗生素極為重要。
輔酶A生化合成路徑中的酵素具有成為新的抗生素藥物之標的的潛力，輔酶A藉由維生素B5經過五個酵素反應步驟所合成，為細胞內重要的乙醯基攜帶者，參與許多生化代謝反應。前人研究指出phosphopantetheine adenylyltransferase (PPAT) 所催化的反應是輔酶A合成路徑中的重要調節步驟，而藉由比對發現在哺乳動物及細菌的同源蛋白間沒有具高相似性的序列，因此PPAT是一個具有開發潛力的抗生素藥物標的。
在前人研究中認為胃幽門桿菌的開放閱讀框(ORF) hp1475編碼著與脂多醣(lipopolysaccharide)合成相關之酵素，然而HP1475蛋白保守性序列指出此基因可能編碼PPAT酵素。在本篇研究中為了更進一步指出目標蛋白的特性，我們選殖了hp1475基因並使其在大腸桿菌中大量表現其產物，而表現所得到的重組單體蛋白HP1475分子量藉由MALDI-TOF測量結果為19.7 kDa，其天然狀態的分子量則藉由膠體過濾層析測定結果為88.39 ± 2.44 kDa，在分析型超高速離心實驗中其分子量為79.86 ± 3.37 kDa，代表HP1475可能以四聚體的形態存在於自然界。本研究藉由核磁共振測定反向酵素活性、並且利用酵素動力學測定Km及Kcat值，確定純化後之重組蛋白具有將dephospho-CoA (dPCoA) 和焦磷酸轉換成4’-posphopantetheine和三磷酸腺苷的能力。藉由圓二色光譜分析HP1475蛋白之二級結構含有63.01 ± 10.04 % α螺旋和3.87 ± 2.40% β摺疊的構造。
將HP1475蛋白序列中具有高度保留性的胺基酸殘基進行點突變，研究結果發現Y7和R88對蛋白的水溶性非常重要。在非還原性不連續蛋白凝膠電泳分析結果中顯示，R88與次單位的組裝相關。藉由pymol軟體預測得知這些具有高度保留性的胺基酸殘基可能位於蛋白質的活性位置並與受質結合能力相關。利用電泳及圓二色光譜分析可得知H18與蛋白的穩定性及二級結構有關。測定反向酵素活性及圓二色光譜分析發現，具有高度保留性的胺基酸殘基對於酵素活性極為重要，進行點突變後會致使蛋白喪失其反向PPAT的活性，而造成活性消失的情形並非因為蛋白質二級結構改變。
我們利用反義股RNA干擾技術研究在活體內HP1475的功能，在大腸桿菌表現系統中反義股RNA干擾造成50%至60%的蛋白抑制效果，而在胃幽門螺旋桿菌中造成 60%的蛋白抑制效果。而此HP1475表現受到抑制的菌株具有減慢生長速率以及細胞內輔酶A堆積的生理現象。

Abstract 5
中文摘要 7
Abbreviations 9
Introduction 11
I. The discovery of Helicobacter pylori 11
II. The characteristics of H. pylori 12
III. Genomic insights into the biology of H. pylori 12
IV. Diseases associated with H. pylori infection 13
V. The antibiotic resistance of H. pylori 13
VI. Coenzyme A (CoA) biosynthesis as a target for antimicrobial drug development 14
VII. The biosynthetic pathway of coenzyme A 15
VIII. The phosphopantetheine adenylyltransferase (PPAT) 16
IX. The study in PPAT structure 18
X. The in vivo function of PPAT 19
XI. hp1475 (CoaD) gene 19
XII. Antisense RNA modulation in H. pylori 20
Materials and methods 23
I. Bacterial strains, plasmids, enzymes and reagents 23
II. Bioinformatic analyses 23
III. Molecular cloning of HP1475 gene 24
IV. Overexpression and affinity purification of HP1475 protein 24
V. Protein electrophoresis, western blotting and immunodetection 25
VI. Determination of kinetic parameters of the reverse reaction of H. pylori HP1475 catalysis 26
VII. The reverse reaction of H. pylori PPAT (HP1475) detected by nuclear magnetic resonance spectroscopy (NMR) 27
VIII. Molecular weight determination of HP1475 protein 27
IX. The determination of native molecular weight of HP1475 by analytical ultracentrifugtion 28
X. Site-directed mutagenesis of HP1475 28
XI. Circular dichroism of HP1475 protein 29
XII. Analysis of HP1475 point-mutated protein in a discontinuous native protein gel electrophoresis system 30
XIII. The effects of point mutations on the reverse reaction of PPAT catalytic activity of HP1475 protein 30
XIV. The construction of hp1475 antisense plasmid 30
XV. Expression of hp1475 antisense RNA in E. coli 31
XVI. The knockdown effect of expression of hp1475 antisense RNA in H. pylori 32
XVII. Determination of coenzyme A 33
Results 34
I. The bioinformation of HP1475 34
II. Molecular cloning of HP1475 35
III. Overexpression and purification of HP1475 protein 35
IV. The steady state kinetic analysis of the reverse reaction catalyzed by HP1475 35
V. The reverse reaction of H. pylori PPAT (HP1475) detected by nuclear magnetic resonance spectroscopy (NMR) 36
VI. Molecular weight determination 37
VII. Circular dichroism of HP1475 protein 37
VIII. Labeling amino acid point mutations of HP1475 in the corresponding positions of the available E. coli PPAT protein structure 38
IX. The effects of point mutations on the expression of HP1475 protein 38
X. Analyses of HP1475 point-mutated proteins in a discontinuous native protein gel electrophoresis system 39
XI. Investigation of the effects of point mutations on the secondary structure of HP1475 by circular dichroism 40
XII. The effects of point mutations on the reverse reaction of HP1475 catalytic activity 40
XIII. The knockdown effect of expression of hp1475 antisense RNA in E. coli 40
XIV. The knockdown effect of expression of hp1475 antisense RNA in H. pylori 41
XV. The growth curve analysis of wild-type H. pylori strain and HP1475 knockdown strains 42
XVI. Determination of the coenzyme A level within wild-type H. pylori strain and HP1475 knockdown strains 42
Discussion 43
References 52
Tables 59
Table 1. Primers used for PCR amplification in this study 59
Table 2. Sequence comparison of HP1475 protein with other bacterial homologues 61
Table 3. Purification of HP1475 protein 62
Table 4. The summary of steady state parameters for HP1475 in the reverse catalytic reaction 63
Table 5. Comparison of biochemical properties of PPAT homologues from different species 64
Figures 65
Figure 1. The structure of coenzyme A 65
Figure 2. The five enzymatic steps of coenzyme A biosynthetic pathway in prokaryotes 66
Figure 3. Molecular cloning of HP1475 67
Figure 4. The reaction scheme of steady state kinetic assay of the reverse reaction of H. pylori HP1475 catalysis 68
Figure 5. Molecular cloning of antisense hp1475 plasmid 69
Figure 6. The bioinformatic information of HP1475 in H. pylori 26695 strain 70
Figure 7. Sequence alignment of the H. pylori PPAT (HP1475) with its homologues 71
Figure 8. Analyses of transmembrane and hydrophobicity profile of HP1475 protein 72
Figure 9. Purification of HP1475 protein with the metal-chelated affinity column using increasing concentrations of imidazole 73
Figure 10. Heterogeneous expression and purification of HP 1475 74
Figure 11. Steady state kinetic analysis of the reverse reaction of H. pylori HP1475 catalysis using dPCoA as the determined substrate 75
Figure 12. Steady state kinetic analysis of the reverse reaction of H. pylori HP1475 catalysis using pyrophosphate as the determined substrate 76
Figure 13. The reverse catalytic reaction of H. pylori PPAT (HP1475) detected by nuclear magnetic resonance spectroscopy (NMR) 77
Figure 14. The subunit molecular weight of H. pylori HP1475 measured by MALDI-TOF mass spectrometry 78
Figure 15. The determination of native molecular weight of HP1475 by gel filtration 79
Figure 16. The determination of native molecular weight of HP1475 by analytical ultracentrifugation 80
Figure 17. The secondary structure of H. pylori HP1475 81
Figure 18. The corresponding positions of amino acid point mutations for HP1475 in the available E. coli PPAT protein structure 82
Figure 19. The effects of point mutations on the expression and the solubility of HP1475 protein 83
Figure 20. Analyses of HP 1475 point-mutated proteins in a discontinuous native protein gel electrophoresis system 84
Figure 21. Investigation of the effects of point mutations on the secondary structure of HP 1475 by circular dichroism (CD) spectra 85
Figure 22. The effects of point mutations on the reverse reaction of HP1475 catalytic activity 86
Figure 23. The prediction of the secondary structure of RNAs expressed from the cloned antisense hp1475 gene fragment 87
Figure 24. The knockdown effect of expression of hp1475 antisense RNA in E. coli 88
Figure 25. The knockdown effect of expression hp1475 antisense RNA in H. pylori 89
Figure 26. The growth curve analysis of wild-type H. pylori strain and HP1475 knockdown strain 90
Figure 27. Determination of the CoA level within wild-type H. pylori strain and HP1475 knockdown strain 91

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