克雷白氏肺炎桿菌CG43第三型線毛用於表面抗原呈現與其表現調控

Abstract

為了可以表現克雷白氏肺炎桿菌第三型線毛的重組質體pmrkABCD來呈列第二型登革熱病毒的套膜蛋白第三區塊（DEN2EIII），首先，我們以此包含主要抗原決定位的核酸為模版，以聚合脢連鎖反應分別增幅可轉譯成約20個胺基酸片段長度的核酸，再將這五段核酸序列分別插入pmrkABCD質體中MrkA A25或D27位置，再將選殖的重組質體包括pA25-1，pD27-1，pD27-2，pD27-3，pD27-4，pD27-5分別轉殖到大腸桿菌JM109。接著，以西方墨點法分別以MrkA和DEN2EDIII多株抗體偵測的結果顯示：雖有不同程度的表現差異，這些重組質體皆可表現帶有DEN2EDIII抗原的重組線毛單體，而此重組的單體蛋白也可組裝成多體的線毛結構；免疫螢光顯微鏡檢分析也確認這些重組的線毛可表現在細菌表面。初步純化結果顯示線毛表現的培養條件仍需改進，只有提升線毛量作為將來免疫小鼠的疫苗來源，才能進一步評估其免疫效果。 大多數的克雷白氏肺炎桿菌臨床分離株帶有可轉錄第三型線毛的基因組，顯示此線毛在克雷白氏肺炎桿菌附著宿主細胞造成感染過程中可能扮演重要的角色。至今，細菌如何調控第三型線毛的表現，仍不清楚。之前的實驗結果顯示，破壞可以調控細菌莢膜產生的調控基因rcsB，會增加第一型纖毛的表現，相反的會降低MrkA蛋白的表現。我們以帶有PmrkA-LacZ報導系統的CG43S3Z01的rcsB缺損株為跳躍子接受株，以X-Gal培養盤篩選顏色變化，在大約70000突變株中挑出八株會改變顏色的突變珠；其中6株會增加LacZ活性，2株會降低LacZ活性。核酸定序分析結果顯示兩株增加LacZ活性的插入點分別是glycosidase和lacI基因，此二基因與LacZ活性調控有關，與MrkA蛋白調控無關。而兩株降低LacZ活性的插入點分別是carbon starvation protein A和會受到滲透壓影響的yehZ基因，此結果確認此系統的選擇效果，但是跳躍子突變效率仍需提高。

We report the use of the type 3 fimbriae, encoded by mrkABCD, from Klebsiella pneumoniae CG43 to display the domain III of envelope protein (E) from type 2 dengue virus, DEN2EDIII. The DNA containing the DEN2EDIII, which has been shown as a major antigenic determinant, was used as a template for PCR amplification to generate 5 DNA segments (~60 bp) encoding different parts of the DEN2EDIII. The PCR products were cloned into the A25 or D27 site on MrkA reside in the plasmid pmrkABCD. The resulting plasmids including pA25-1, pD27-1, pD27-2, pD27-3, pD27-4 and pD27-5 were then transformed separatelyinto E. coli JM109. The analysis of these recombinant type 3 fimbriae assessed with western blotting hybridization against anti-MrkA or anti-DEN2EDIII antibody confirmed the expression of the recombinant proteins, although with different levels. And the recombinant subunits could assemble into polymeric fimbriae from monomers. The analysis of immunofluorescence microscopy using anti-MrkA or anti-DEN2EDIII antibody also demonstrated the expression of the recombinant type 3 fimbriae on the surface of the transformed bacteria. However, culture condition and purification method have to be improved to obtain sufficient amount of the recombinant fimbriae. The subsequent use as the vaccine antigen for mouse immunization and the efficacy could then be evaluated. The fact that most Klebsiella pneumoniae clinical isolates carry type 3 fimbriae gene clusters and hence play an important role as a major adhesin for the bacterial attachment to the host cell to establish an infection has been speculated. Nevertheless, how the expression of the type 3 fimbriae controlled remains unknown. Herein, a Tn5 mediated mutagenesis was employed to K. pneumoniae CG43Z01rcsB- strain carrying Pmrk-lacZ. Eight out of approximately 70000 Tn5-insertion mutants showed alteration of LacZ activity and were isolated. Six mutants exhibited an increased LacZ activity and two mutants carried a decreased LacZ activity. After the inserted were regions isolated and the sequences determined, it was revealed that the disrupted genes include the gene encoding a putative glycosidase and lacI for the mutants with increasing LacZ activity, and the gene encoding carbon starvation protein and an osmolarity-inducible protein YehZ. The results demonstrated the feasibility of the reporter-selection and the efficiency of Tn-mutagenesis have to be improved, however.