克雷伯氏肺炎桿菌預測溶菌酶之選殖與功能分析

Abstract

比對克雷伯氏肺炎桿菌(Klebsiella pneumoniae) NTUH-K2044菌株與MGH 78578菌株之DNA序列，發現在NTUH-K2044全基因體序列之2089556至2114933鹼基對(base pairs)位置(約25 kb)與MGH 78578菌株之序列有很大的差異。以National center for biotechnology information Basic Local Alignment Search Tool (NCBI BLAST)生物資訊工具分析此段序列，發現其中有一開放讀架(open reading frame)預測為溶菌酶(endolysin)基因。溶菌酶為噬菌體(phage)進行溶菌期(lytic cycle)所需之酵素，其能作用在細菌之肽聚醣(peptidoglycan)而使細菌之細胞壁分解，以利組裝好的噬菌體釋出。先前的研究報告指出，使用外加溶菌酶的方式作用於細菌，能夠使細菌細胞壁分解而殺死之，這也使得溶菌酶具有作為替代性殺菌劑的發展潛力。本實驗將此預測之溶菌酶基因轉殖於大腸桿菌（Escherichia coli），進一步表現與純化此蛋白質。將此蛋白質作用於不同細菌，測試作用不同時間下的細菌存活率。結果顯示，以蛋白質50微克(μg)作用於大腸桿菌15分鐘，即能降低細菌存活率至7.22 %。另外，此蛋白質的殺菌效力具有選擇性，受測菌株中，對於大腸桿菌的殺菌效力最佳，其次為克雷伯氏肺炎桿菌，對於枯草桿菌（Bacillus subtilis）效果較差。為了更進一步確認此蛋白質作用的受質為肽聚醣，因此將蛋白質加入肽聚醣混濁液進行肽聚醣分解實驗（peptidoglycan degrading assay），結果發現此蛋白質無法對枯草桿菌之肽聚醣進行分解。推測此蛋白質的殺菌效果，可能是針對細菌其他的部分作為標的。

Comparison of the genome sequences of Klebsiella pneumoniae NTUH-K2044 strain with those of MGH 78578 strain, we found that there is a different region (about 25 kb) in the 2089556 to 2114933 sites of the genome of NTUH-K2044 strain. Analysis of the different region by using bioinformatics tools, National center for biotechnology information Basic Local Alignment Search Tool (NCBI BLAST), we found that there is an open reading frame being predicted as an endolysin gene. Endolysin is the enzyme encoded by bacteriophage and directly targets bonds in the peptidoglycan layer of the bacterial cell wall. The result of this activity is degradation of the rigid murein layer and release of newly assembled virions at the terminal stage of the phage reproduction cycle. According to previous reports, the capability of endolysin to digest the cell wall when applying exogenously to bacterial cells has enabled its use as alternative antibacterials. The putative endolysin gene was clone to Escherichia coli, and protein was expressed and purificated. And then we applied the purified protein exogenously to bacterial cells, and their survival rate was observed. The results showed that 50 μg purified protein can decrease the survival rate of Escherichia coli to 7.22% in 15 minutes. On the other hand, the protein also showed selective bactericidal activity. Among the tested strains, Escherichia coli is the most sensitive to the protein, Klebsiella pneumoniae is the second, and Bacillus subtilis is the least sensitive to the putative endolysin. In order to find out if peptidoglycan is the substrate of the protein, we performed peptidoglycan degrading assay. The results showed that the protein can not degrade peptidoglycan of Bacillus subtilis. It may be other components of bacteria that the protein targets, and results in the bactericidal effect.