臺灣博碩士論文加值系統

鮑氏不動桿菌 (Acinetobacter baumannii)是一種伺機性致病菌，在大量使用抗生素治療的環境下，其變成多重抗藥性的鮑氏不動桿菌 (multidrug- resistant Acinetobacter baumannii, 簡稱MDRAB)的機率與日俱增，已然嚴重威脅全球人類的健康。因此，發展一種可以有效預防MDRAB感染的方案目前在臨床上是相當迫切的。其中，利用致病菌上的表面多醣(exopolysaccharide)或其寡糖片段和載體蛋白(carrier protein)，透過特定的連接子(chemical linker)連結在一起所製成的醣複合疫苗(glycoconjugate vaccine)已被證實在預防其他致病菌的感染具有相當優異的效果，像是肺炎鏈球菌，腦膜炎雙球菌…等等。然而，在之前文獻認為，和用全長的表面多醣比較，利用表面寡醣片段架接在載體蛋白能夠引發更為顯著的免疫反應。於是，我們希望透過在噬菌體(bacteriophage)的尾部刺突蛋白(tail spike protein)具有醣水解酶的特性，獲得均質的細菌表面寡糖片段，以提升針對MDRAB的醣共軛疫苗的成效。而在之前的文獻提出在台灣有篩選到兩株不同會感染鮑氏不動桿菌的噬菌體，ΦAB6和ΦAB2，分別感染不同宿主Ab-54149和Ab-SK44。因此在本研究中，我們表現了這兩株噬菌體的重組尾部刺突蛋白，ΦAB6 TSP和ΦAB2 TSP，並且確認了其具有分別水解Ab-54149和Ab-SK44表面多醣的活性。而藉由這兩種尾端蛋白的產物，可以解析出Ab-54149和Ab-SK44表面多醣的結構。Ab-54149 表面多醣的結構和之前所報導的兩株具有抗藥性鮑氏不動桿菌上的表面多醣結構是一樣的，而Ab-SK44的表面多醣則可能和保護菌株避開補體(complement)的攻擊有關。另外，也得知ΦAB6 TSP水解的產物主要是由含有兩個pseudaminic acid的兩個重複單元(repeat unit)的寡醣所組成，而ΦAB2 TSP水解的產物則是只有一個重複單元的寡醣所組成。而利用這兩種表面多醣誘發所產生的抗體，對於其他在台灣臨床鮑氏不動桿菌株的表面多醣具有相當良好的識別率，因此這兩種表面多醣相當適合做為發展醣共軛疫苗的原料。於是，我們將兩種經由尾端蛋白水解過後的寡醣片段架接到載體蛋白上，並將此複合物注射到兔子身上，發現具有很好的免疫原性(immunogenicity)，可以再更進一步作活體的測試。另外，我們也針對ΦAB6 TSP去做其結構和水解機制的探討。ΦAB6 TSP是由三個以β-helix為主體的單元聚合在一起的結構，而其活性位點(active site)則是位於每兩個β-helix的中間介面所形成的凹槽中。在活性位點中的麩氨酸(glutamic acid)425和447會經由維持還原端位向的機制將多醣進行水解。再者，Pse則是被認為在ΦAB6 TSP辨認受質的過程中便演相當重要的角色，也可能是造成ΦAB6 TSP具有高度受質專一性的原因。本研究提供了有關未來利用改造尾端蛋白來獲取更均質，更多樣化，更有免疫活性的表面寡醣片段的資訊，這對發展對抗MDRAB的醣複合疫苗將會是一大幫助。

With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines (GCV), made up of bacterial exopolysaccharide (EPS) and carrier protein conjugating with chemical linker, are an emerging therapeutic to combat bacterial infection. However, some previous literatures demonstrated that carbohydrate epitopes bearing proper repeat units of bacterial polysaccharide usually induced stronger immune responses in the vaccination process than those using whole polysaccharide. Therefore, we hopefully acquire the homogenous oligosaccharide fragments via bacteriophage tail spike protein (TSP) carrying glycohydrolase activity. Herein, we characterized that the bacteriophage ΦAB6 and ΦAB2 tail spike protein (TSP) enable to hydrolyze specifically the EPS of A. baumannii strain 54149 and SK44 respectively (Ab-54149 and Ab-SK44). Based on structural analysis of TSP-digested product, Ab-54149 EPS exhibited the same chemical structure as that of two antibiotic-resistant A. baumannii strains, and Ab-SK44 EPS might protect bacteria from complement-mediated killing. The ΦAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). By contrast, ΦAB2 TSP-digested product contained only one repeat unit. The antibodies induced by whole EPS of Ab-54149 and Ab-SK44 were capable of recognizing a number of A. baumannii clinical strains EPS. For potential use in GCV, both TSP-digested products were further conjugated to carrier protein CRM197 and subsequently injected into rabbits. The boosted serum from rabbits could recognize the whole extract and digested product of Ab-54149 and Ab-SK44 EPS respectively, indicated high immunogenicity of conjugate complex. Notably, both boosted serum displayed significantly better sensitivity toward whole EPS. The 1.48-1.89-Å resolution crystal structures of an N-terminally-truncated ΦAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric β-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for ΦAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of GCV against A. baumannii infection.

謝誌....................................................i
中文摘要................................................ii
英文摘要................................................iv
目錄...................................................vi
Abbreviations..........................................x
List of Figures………………………………………………………………………….xi
Chapter 1: Introduction………………………………………………………………..1
1-1. General introduction of Acinetobacter baumannii……..1
1-2. Emergence of multi-drug resistant Acinetobacter baumannii…………………….......................................2
1-3. Virulence factors for Acinetobacter baumannii pathogenicity…………………....................................4
1-4. Phage and its tail spike protein………………………………………....6
1-5. Glycoconjugate vaccine………………………………………………………….......8
1-6. Specific Aims…………………………………………………………………............10
Chapter 2: Materials and Methods…………………………………………………....12
2-1. Protein expression and purification…………………………………..12
2-2. Extraction of bacterial surface polysaccharides...13
2-3. Digestion of A. baumannii surface polysaccharide by ΦAB6 TSP……………..........................................14
2-4. Top agar assay of enzyme activity……………………………………...14
2-5. Structure determination of Ab-54149 surface polysaccharide by NMR..................................15
2-6. Mass spectrometry analysis of digested products of Ab-54149 surface polysaccharide………………………………………………………………16
2-7. Enzyme kinetic assay…………………………………………………………........16
2-8. Antiserum production and dot blot assay………………………..17
2-9. Crystallization and X-ray data collection………………………18
2-10. Structure determination and refinement……………………………20
2-11. Analytical ultracentrifugation analysis……………………..21
Chapter 3: Results
3-1. Characterization of ΦAB6 TSP and ΦAB2 TSP…………………….22
3-1-1. Glycosidase activity of ΦAB6 TSP and ΦAB2 TSP………22
3-1-2. Optimized condition for enzymatic activity of ΦAB6 TSP andΦAB2 TSP….......................................24
3-1-3. Further characterization of ΦAB6 TSP………………………………26
3-2. Structure of EPS of A. baumannii strains 54149 and SK44……………………...........................................27
3-2-1. Structure of EPS of A. baumannii strain 54149…..27
3-2-2. Structure of EPS of A. baumannii strain SK44……….30
3-3. The 3D crystal structure of ΦAB6 TSP……………………………………32
3-3-1. Overall structure of ΦAB6 TSP∆N………………………….………...32
3-3-2. Structure of ΦAB6 TSP∆N monomer……………………………………...33
3-3-3. Structure of ΦAB6 TSP∆N trimer…………………………………………….36
3-4. The complex structure of ΦAB6 TSP………………………………………….40
3-4-1. Structure of carbohydrate-binding groove……………………40
3-4-2. Catalytic center of ΦAB6 TSP………………………………………………..43
3-5. Immunogenicity of glycoconjugate complex…………………...47
3-5-1. Clinical survey of EPS induced antibody…………………..47
3-5-2. Synthesis of glycoconjugate complex…………………………………48
3-5-3. Dot blots analysis of anti-serum which were individually produced by two glycoconjugate comprising of ΦAB6 TSP and ΦAB2 TSP digested product………………………………………..51
Chapter 4: Conclusion and Discussion…………………………………………….…53
4-1. Exopolysaccahride of Ab-54149 and Ab-SK44………………………54
4-2. Structural comparison of phage TSPs…………………………………..55
4-3. Potential application of ΦAB6 TSP complex structures.............................................59
4-4. Serum boosted by glycoconjugate…..…………………………………………61
Chapter 5: Perspective……………………………………………………………….........63
Tables…………………………………………………………………………………..................64
Table 1. Data collection and refinement statistics………..64
Table 2. The proton (1H) and carbon (13C) chemical shifts on NMR spectra of ΦAB6 TSP-digested products of Ab-54149 surface polysaccharide……………............................65
Table 3. Kinetic data of wild-type and mutant ΦAB6 TSPs...................................................65
Table 4. The population of extracted EPS from 250 clinical A. baumannii strains recognized by Ab-54149 or Ab-SK44 EPS induced antibodies……...…………................66
Acknowledgements…………………………………………………………………….............67
References……………………………………………………………………………................68
Appendix……………………………………………………………………………….................77

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