分離K47莢膜型克雷伯氏肺炎桿菌之噬菌體與其莢膜分解酵素功能分析

Abstract

在人類和致病菌的戰爭中，抗生素一直是最有力的武器，然而抗生素的濫用使得臨床上有越來越多多重抗藥性的菌株被發現，其中克雷伯氏肺炎桿菌（Klebsiella pneumoniae）以其厚重的莢膜與不俗的生物膜生成能力，造成棘手的院內感染，K47莢膜型（Capsular types）的克雷伯氏肺炎桿菌更是攻破目前抗生素最後防線的碳青黴烯（Carbapenem），發展出抗藥性。噬菌體（Bacteriophage）作為細菌最古老的宿敵，相較於抗生素有著專一性殺菌、迭代速度快、能夠清除生物膜等優點。在抗藥性菌株越發氾濫的情況下，以噬菌體進行殺菌的噬菌體療法（Phage therapy）無疑是個優秀的替代方案。在針對克雷伯氏肺炎桿菌的部分，有些噬菌體可以透過自身帶有的專一性莢膜分解酵素（Capsule depolymerase）突破菌株生物膜與莢膜，達成入侵宿主的目標。 本研究首先從環境廢水中純化出針對K47莢膜型的噬菌體，並透過點試驗檢測宿主範圍、觀察單一型態溶菌斑的雙圈呈現情形以及限制性片段長度多態性驗證初步判定是否與其它噬菌體相同，找到能感染多種K47菌株，且具有明顯雙圈溶菌斑的噬菌體ΦK47_1和ΦK47_4。後續於毒殺試驗與抗生物膜試驗中，確認了ΦK47_1和ΦK47_4皆有不錯的殺菌效果以及優秀的生物膜清除能力。於是將ΦK47_1和ΦK47_4基因體萃取出來後進行全基因體定序。在與美國國家生物技術資訊中心資料庫比對後，於ΦK47_1中找到可能編碼具有莢膜分解酵素活性蛋白的CDS_001、CDS_003、CDS_005、CDS_007與CDS_008，以此五段基因為模板進行基因重組蛋白的表現。其中序列比對為尾刺突蛋白（Tail spike protein）的CDS_003表現出來的重組蛋白Tsp03，能在對K47菌株產生溶菌斑，且能有效降解多醣。期望未來能進一步分析Tsp03與莢膜多醣反應的結構，以利進一步開發接合型疫苗（Conjugate vaccine）。

Antibiotics are the most powerful weapon in the war between humans and pathogenic bacteria. However, due to the misuse of antibiotics, more and more multi-drug-resistant strains have been discovered clinically, including Klebsiella pneumonia. This bacterium is famous for causing troublesome hospital-acquired infections with its thick capsule and excellent biofilm-generating ability. Moreover, the clinical data suggests that K47 capsular type K. pneumoniae gain carbapenem-resistant genes. This means that the antibiotics used as the last line of defense are no longer fully effective against K. pneumoniae. Therefore, it is imperative to find new treatments.Bacteriophages are the oldest enemies of bacteria. Compared with antibiotics, they have the advantages of specific targeting, fast iteration speed, and the ability to clear bacterial biofilms. As drug-resistant strains become increasingly prevalent, phage therapy, which uses phages to kill bacteria, is a brilliant alternative. For K. pneumoniae, some phages can break through the biofilm and capsule through their own specific targeting capsule depolymerases. This study first purified phages targeting the K47 type strains from environmental sewages. Next, phages were chosen based on whether they presented double-zone plaques and were tested for host range through spot tests. Restriction fragment length polymorphism (RFLP) was conducted to preliminarily determine whether they were the same as previously discovered phages. ΦK47_1 and ΦK47_4 were found, and their plaques presented obvious double zones. Killing assays and anti-biofilm tests confirmed that both ΦK47_1 and ΦK47_4 had good bactericidal effects and excellent biofilm removal capability. Therefore, the ΦK47_1 and ΦK47_4 genomes were extracted and subjected to whole genome sequencing. The sequencing data were aligned with the National Center for Biotechnology Information (NCBI) database. The results showed that five genes, CDS_001, CDS_003, CDS_005, CDS_007, and CDS_008, might have coded for proteins with capsule depolymerase activity. Hence, these five gene segments were selected as templates to express recombinant proteins. Among them, the recombinant protein Tsp03 produced lytic plaques on K47 strains in the spot test and effectively degraded capsular polysaccharide. The structure of the reaction between Tsp03 and capsular polysaccharide could be further analyzed in the future to facilitate the development of a conjugate vaccine.