以噬菌體重組基因技術探討多宿主噬菌體K11-7-1-1之莢膜分解酵素活性

Abstract

克雷白氏肺炎桿菌為腸內菌科的格蘭氏陰性桿菌，莢膜為重要致病因子，並且與克雷白氏肺炎桿菌所引起的感染侵襲力有關，因此正確鑑定克雷白氏肺炎桿菌之莢膜型具臨床重要意義。傳統使用抗血清分辨莢膜型，然而血清學鑑定方法的敏感度及專一性不佳，為有效進行莢膜分型，本實驗室嘗試發展噬菌體醣類分解酵素分型系統。 噬菌體感染具莢膜細菌的過程中，需要莢膜分解酵素輔助降解莢膜，而我們自原水分離的噬菌體K11-7-1-1可辨認克雷白氏肺炎桿菌丹麥標準菌種K1，K11，K21，K25，K30，K35，K64及K69共8種莢膜型，推測噬菌體K11-7-1-1應具有莢膜分解酵素，而定序其基因體序列後比對到10個與莢膜分解酵素相似之預測基因。為探討預測基因實際功能，首先以大腸桿菌蛋白質誘導系統表現蛋白質並測試活性，3個預測基因S1-1、S2-7、S2-2表現出蛋白質活性，S1-1和S2-7對應至莢膜型K11，而S2-2則對應莢膜型K25。接著以建構噬菌體基因剔除突變株方式，分析噬菌體K11-7-1-1中可能的莢膜分解酵素基因之確實功能以及莢膜型活性，得到對應至K35的S2-3及對應至K30及K69莢膜型的S2-6共兩個基因剔除株，仍有5個預測基因的功能未知。 噬菌體無標記基因剔除技術的建立，可以應用於研究噬菌體中可能為莢膜分解酵素基因，幫助確認莢膜分解酵素的活性，另外我們嘗試建造基因置換突變株，雖然目前未得到置換入外來酵素基因的突變株，但透過成功建造CRISPR之間置序列基因置換突變株，顯示基因置換株的可行性，未來可期盼基因置換株成為研究基因功能的另一方式。

Klebsiella pnuemoniae, a Gram-negative bacillus, belongs to Enterobacteriaceae. Capsule is one of virulence factors, and researches have implied that capsular type contributes to severity of disease. Thus, how to rapidly and correctly identify the capsular type of K. pneumoniae is a clinically significant issue. Serological technique is the frequently used method in the laboratory, while further tests have to be practiced to improve the sensitivity and specificity. We attempt to establish a typing system using purified capsular depolymerases to assist diagnosis in our laboratory, and it is leading to determine the activity of putative enzyme. During infection of encapsulated bacteria, the phages secrete endoglycosidases to degrade capsule where the primary receptor buried in order to attach the primary receptor. We isolated a new phage, named as φK11-7-1-1, which infects Denmark reference strain K1, K11, K21, K25, K30, K35, K64 as well as K69. There are 10 open reading frames (ORFs) expected to be capsular depolymerases according to sequence similarity. These putative enzymes were expressed in Escherichia coli to study the activity, S1-1 as well as S2-7 were shown to be the specific enzymes to K11, and S2-2 exhibited its activity to K25. We then constructed unmarked deletion of individual putative enzymes in φK11-7-1-1 as a mean to study functions. After construction of recombinant phages, S2-3 and S2-6 deletion mutants were obtained. S2-3 and S2-6 were separately revealed to be specific to K35, both K30 and K69. There are five putative enzymes remained unidentified function. The technique of constructing recombinant phages may be aid of and applied to study putative enzymes or gene functions in phages. We also tried to substitute a foreign enzyme gene, which ends in vain, but a replacement mutant was built by introducing a 33-nucleotide CRISPR spacer sequence, indicating that replacement mutants might be practicable as another model studying enzyme activity.