克雷白氏肺炎桿菌NTUH-K2044 中纖毛黏附蛋白KpfD的功能性分析

Abstract

纖毛在細菌感染時扮演黏附宿主細胞的重要角色。基因體解碼後，序列分析顯示細菌基因體中可比對出多套表現纖毛的基因組，在克雷白氏肺炎桿菌NTUH-K2044中，除了第一、三型纖毛基因組外，也發現其他未曾被報導過的七套纖毛基因組，分別被命名為kpa、kpb、kpc、kpd、kpe、kpf 及 kpg纖毛。先前實驗室建立第三型纖毛表現系統，並以此系統表現MrkD以外的黏附蛋白，藉以探討這些未知黏附蛋白的活性。本論文除了以免疫螢光標定証實此重組纖毛結構蛋白的表現外，還以血球凝集試驗確認KpfD凝集天竺鼠紅血球的能力，及可被0.002 M甘露醣所抑制凝集作用的黏附特性。同時，我們構築可表現重組KpfD蛋白的質體，在大腸桿菌中大量表現並純化KpfD重組蛋白來製備多株抗體，進一步利用此專一之KpfD抗體藉西方點墨實驗及免疫螢光分析，証實黏附蛋白KpfD可以成功的表現於重組纖毛成頂端。接著，我們利用醣類抑制實驗證實KpfD黏附蛋凝集天竺鼠紅血球的活性可被0.001 M甘露醣抑制，其凝集活性也可被0.01 M 的 N-acetyl-D-glucoseamind抑制卻不受D-fucose的影響；而利用KpfD專一性抗體證實此凝集現象確實經由KpfD的黏附作用。為了了解KpfD與甘露醣交互作用的區域，我們比較分析KpfD與第一型纖毛黏附蛋白FimH的二級與三級結構，預測KpfD與甘露醣交互作用的區域可能在KpfD D73至A83之間，因此，我們分別表現並純化包含及不含此預測作用區域的KpfD截短蛋白，然而，血球凝集競爭試驗顯示兩者皆無法抑制凝集現象。細胞黏附能力測試顯示表現重組第三型纖毛的大腸桿菌具有自我凝集(autoaggregation)的能力，因此，我們建構表現kpf纖毛的質體pKpfABCD及pKpfRABCD，而利用KpfD抗體進行西方點墨法及免疫螢光分析，證明pKpfRABCD可以使大腸桿菌表現kpf纖毛；血球凝集測試也證實帶有pKpfRABCD質體的大腸桿菌具有凝集天竺鼠血球的活性，而且此凝集活性可受到甘露醣抑制。

Fimbriae play an important role in establishing an infection by adhering to specific host tissues. The available genome sequences of many enterobacteria reveal multiple fimbrial loci are commonly contained in a genome. In addition to type 1 and 3 fimbrial gene clusters, seven novel fimbrial operons were identified in Klebsiella pneumoniae NTUH K2044 and named kpa, kpb, kpc, kpd, kpe, kpf and kpg. We have previously generated a type 3 fimbrial display system by replacing the mrkD adhesin gene with each of the fimbrial adhesion genes. In the study, analysis using immuno-fluorescence microscopy (IFM) and western blot hybridization were employed to demonstrate the recombinant type 3 fimbriae expressed properly. The recombinant fimbriae was found to be able to bind to erythrocytes of guinea pig, and the hemaaglutination (HA) activity was inhibited by 0.002 M D-mannose. In addition, the recombinant plasmid for overexpression of KpfD was generated and transformed into Escherichia coli NovaBlue (DE3). The heterologous expressed KpfD protein was then purified for polyclone antibody preparation. The antibody was then used to demonstrate the KpfD was assembled on the tip of the recombinant type 3 fimbriae by western blot and IFM analysis. The HA competition analysis revealed that the agglutination activity could be inhibited by 0.001 M D-mannose or 0.01 M N-acetyl-D-glucoseamind, but not affected by D-fucose. Furthermore, the KpfD antibody could inhibit the HA activity indicating KpfD adhesin is responsible for the specific binding activity to erythrocytes of guinea pig. Comparative analysis of the secondary and tertiary structure with E. coli FimH revealed a putative mannose binding pocket on KpfD D73 to A83.The recombinant plasmids containing truncation of the KpfD protein with or without the predicted mannose binding pocket were generated and the recombinant proteins purified for competition assay of the HA activity. However, neither recombinant protein could inhibit the HA activity. Cell adherence analysis indicated that the overexpression of the type 3 fimbriae on the surface of the recombinant E. coli rendered an autoaggregation phenotype. In order to prevent from the overestimated non-specific cell binding activity, the recombinant plasmid containing kpfABCD or kpfRABCD was generated and the expression of the recombinant kpf fimbriae was assessed by western blot hybridization, IFM assay, and HA activity measurement. The results indicated that the recombinant E.coli expressed kpf fimbriae on the surface exerted an HA activity that could be inhibited by D-mannose.