具有莢膜專一性之克雷白氏肺炎桿菌噬菌體基因體分析及其重組噬菌體之建構

Abstract

中文摘要 克雷白氏肺炎桿菌是革蘭氏陰性桿菌，莢膜為重要的致病因子之一。傳統上是利用血清法來進行莢膜分型，然而血清分型法敏感度與專一性都不佳，因此先前有研究利用噬菌體來進行克雷白氏肺炎桿菌的部分莢膜分型，發現可感染克雷白氏肺炎桿菌的噬菌體，尾端通常帶有可分解宿主莢膜的醣類分解酵素(Capsule depolymerase)，然而先前研究並無核酸序列或病毒檢體留存。因此本實驗室重新研究使用噬菌體所帶有的莢膜醣類分解酵素來區分莢膜型的可能。本實驗室目前已成功建立以噬菌體進行莢膜分型系統，並發現若以噬菌體之莢膜醣類分解酵素進行分型，可增加特異性和敏感性。本實驗室在水源中分離出的噬菌體Ref-K13-1，透過高通量定序，完成了Ref-K13-1的基因體定序，經過基因比對，Ref-K13-1的ORF2和ORF3分別比對到putative EPS depolymerase wceF precursor和endosialidase。判斷是莢膜醣類分解酵素的基因片段，並且初步先在大腸桿菌的表現系統表現莢膜醣類分解酵素，表現的結果發現ORF2和ORF3都不具活性，且ORF3懷疑有被切割的情形。以大腸桿菌蛋白質表現純化系統，常發現這些酵素不表現、溶解度差、被切割和沒有功能。因而嘗試利用噬菌體的基因重組方式，建構剔除Ref-K13-1_ORF3的噬菌體重組株，希望藉由可感染宿主的改變以推測酵素基因的功能，由於無法挑到單一重組株，因此懷疑ORF3是複製的必要基因。因此，建構Ref-K13-1_ORF3預計酵素C端加上His-Tag之重組噬菌體，預期以噬菌體大量表現蛋白質，目前已成功分離酵素基因之重組噬菌體，進而嘗試從預計酵素基因重組之噬菌體純化出蛋白質，但目前無法偵測到帶有His-tag重組蛋白質，懷疑是在蛋白質組裝過程，C端經過修飾而使得His-tag無法被偵測。更進一步建立了Ref-K13-1_ORF3之N端加入His-Tag的重組噬菌體，卻無法挑到單一的重組噬菌體。推測為在N端加入His-Tag不能順利組裝成重組噬菌體。因此本研究已初步成功建立噬菌體的重組方法，雖然目前無法用來純化與表現醣解酵素，未來期待可應用在噬菌體的基因表現與功能研究。

Abstract Klebsiella pneumoniae is a Gram negative and rod shaped bacteria. Capsule is one of the important virulent factors. Traditionally, the method for determination of K. pneumoniae capsular type is serotyping, but the sensitivity and specificity of capsule serotyping are not good enough. Previous studies have shown that bacteriophages which infected K. pneumoniae can be used for determining part of capsule types and often carried the capsule depolymerase on the tail fiber or tail spike. However, those phages were no longer available and there was no sequencing result. Now, a capsule typing system by using bacteriophages/capsule depolymerases was established in our lab. We found that the sensitivity and specificity for capsule typing are better by using the capsule depolymerases than by using bacteriophages. We isolated the phage Ref-K13-1 which infected capsular type K2 and K13 strains from water. After annotation of the genome sequences of phage Ref-K13-1, we found orf2 encodes for putative EPS depolymerase wceF precursor and orf3 encodes for endosialidase. At first, we expressed and purified recombinant ORF2 and ORF3 in E. coli. But both of them did not revealed activities for depolymerization of K2 or K13 capsules, moreover, ORF3 even have been cleavaged. Therefore, we tried to establish the method for generation of recombinant phages by using homologous recombination. At first, we try to generate orf3 deletion recombinant phage, in order to see whether the host range will be changed or not. However, the orf3 deletion phage could not be obtained by massive screening of phage plaque after homologous recombination. This result suggested that orf3 is an essential gene for phage infection. Meanwhile, we try to add C terminal His tag after ORF3 for further protein purification. The recombinant phage whose 3’end of orf3 was added by the His-tag sequences was successfully generated. But the His-tag protein cannot be detected after protein purification. We suggest that C terminal His-tag might be cleavaged. Hence, we try to add His-tag to the N-terminal of ORF3. But we cannot get the recombinant phage after screening. The result suggested that ORF3 with N terminal His-tag might not be assembled to the phage particle correctly. In conclusion, although we cannot express recombinant endoglycosidase currently, we have successfully established the method for generating recombinant phages in K. pneumoniae. This recombinant method can be used for the study of the phage genes in the future.