2011-05-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642542摘要：克雷伯氏肺炎桿菌所引起的社區型肝膿瘍合併腦膜炎以及眼內炎已成為全球新興感染症，已有研究顯示細菌致病力與其莢膜型相關，其中莢膜型K1 與克雷伯氏肺炎桿菌所引起的社區型肝膿瘍最為相關，其次為K2。除此之外，血清型K5、K20、K54，K57以及一新莢膜型N1 菌株似乎也有相關性。由於多醣體與攜帶蛋白質結合之疫苗可提升T 細胞對醣類產生的免疫抗原性(T-cell dependent immunogenicity )，因此發展建構以K1以及K2 等莢膜多醣體與蛋白質接合的疫苗，應是較安全並且有效的預防克雷伯氏肺炎桿菌引起肝膿瘍的方法。已知菌株莢膜為聚合多醣，分子相當巨大，必須將莢膜多醣體降解成較小分子才能有效接合至蛋白質，而常用的化學處理方法雖然可將莢膜降解，但都同時會破壞其修飾基團而喪失莢膜抗原性，為了建立克雷伯氏肺炎桿菌莢膜接合疫苗發展有效的方法，本實驗室嘗試由未處理過之原水分離只感染K1 莢膜型菌株的噬菌體，且發現直接以噬菌體和莢膜多醣體反應，莢膜確實被降解而且不破壞修飾，因此這個噬菌體應該具有特異的醣類降解酶。本子計劃將負責分離可感染造成社區型肝膿瘍特異莢膜型之噬菌體，且進一步找出可分解克雷伯氏肺炎桿菌莢膜之特異的醣類降解酶。將由吳世雄教授實驗室(子計劃二)詳細分析酵素活性與功能，以這些醣類降解酶分解莢膜多醣體將可控制適當的酵素反應條件使莢膜分解成最適合接合的長度。並由任建台博士、吳宗益教授實驗室將降解之莢膜寡醣和蛋白質接合成疫苗(子計劃三)。此外，本實驗室已於先前的研究中建立了一SPF (BALB/c)小鼠動物模式，將克雷伯氏肺炎桿菌以腹腔注射或胃內注射的方式感染小鼠，可使小鼠產生菌血症、肝膿瘍、腦膜炎以及眼內炎等在受到克雷伯氏肺炎桿菌感染之病患所見的病徵。因此於本子計畫中我們將先以此動物模式評估不接合攜帶蛋白質之莢膜多醣體疫苗之效能，再以此動物模式評估一般攜帶蛋白質或克雷伯氏肺炎桿菌高抗原性特異蛋白接合之莢膜多醣體疫苗的安全性及其效用。成功發展之疫苗將不僅用於預防克雷伯氏肺炎桿菌所引起的肝膿瘍，未來也可延伸應用於特異莢膜型克雷伯氏肺炎桿菌造成的其他疾病。<br> Abstract: Community-acquired pyogenic liver abscess (PLA) caused by K. pneumoniae complicatedwith metastatic meningitis and endophthalmitis has emerged globally. The capsular typeswere shown to be associated with bacterial virulence. K1 capsular type was the most stronglyassociated with K. pneumoniae causing PLA, and capsular type K2 was the second. Inaddition to K1, K2, K. pneumoniae strains causing PLA seemed to be also associated withcapsular types K5, K20, K54, K57, and a new type (named as N1 type). Conjugating surfacepolysaccharides to carrier proteins enhances the T-cell dependent immunogenicity of thepolysaccharides. Therefore, construct a capsular polysaccharide (CPS)-protein conjugatevaccine would be safer and more efficient for prevention of K. pneumoniae causing PLA. Thebacterial capsular polysaccharide is a huge polymer. Depolymerization of CPS will increasethe efficiency to conjugate with protein. However, all the current chemical methods cannotdepolymerize the CPS without disrupting the immunogenicity of CPS due to removal ofcapsular modifications. We have tried to isolate the bacteriophages which infected K1capsular type strains from untreated water. This phage was characterized and showed todepolymerize CPS without interference of modifications. Therefore, the isolated phage shouldhave the CPS-specific endoglycosidase(s). In this component project, we will isolate at least 7specific bacteriophages which infect K. pneumoniae causing PLA and further identify theirendoglycosidases for capsules of K. pneumoniae causing PLA. Dr. Shih-Hsiung Wu(subproject 2) will be responsible for determination of the kinetics and property of enzymes.Treatment condition of endoglycosidases can be tested and the optimal reaction conditions fordepolymerization will be deterimnied. The depolymerized capsule with or without proteinconjugation will be generated by Dr. Chien-Tai Ren and Dr. Chung-Yi Wu’s laboratory(subproject 3). Also, we have developed a SPF (BALB/c) mice model that mice infected withPLA K. pneumoniae strain intraperitoneally or intragastrically exhibited bacteremia, PLA,meningitis, and endophthalmitis. In this component project, we will use the PLA mice modelto evaluate the safety and protective efficacy of the polysaccharide-only vaccine as wellas CPS-conjugated vaccines (conjugated with approved carrier proteins or K. pneumoniaespecific immunodominant proteins). The CPS based vaccine(s) will be not only used forprevention of pyogenic liver abscess, but also other diseases caused by some specific capsulartypes of K. pneumoniae in the future.克雷伯氏肺炎桿菌肝膿瘍莢膜攜帶蛋白質噬菌體醣類降解酶疫苗Klebsiella pneumoniaeliver abscesscapsulecarrier proteinsbacteriophageendoglycosidasevaccine