利用黏菌模式發現克雷伯氏肺炎桿菌抗吞噬作用相關的致病基因

Abstract

吞噬作用是宿主對抗致病微生物的第一道防線，因此，對於防禦細菌入侵扮演重要的角色。而在細菌中同樣也有重要的致病基因負責抵禦吞噬作用，進而成功的感染人體。克雷伯氏肺炎桿菌(Klebsiella pneumoniae)引起之化膿性肝膿瘍為台灣重要之新興感染症，其致病機制目前所知和先天免疫中之血清與吞噬抗性最相關。本實驗室原來利用黏性去篩選一突變株庫，結果得到一致病基因為magA，後來證實是負責K1莢膜多醣體合成的重要基因。由於用黏性之篩檢侷限於莢膜與外壁相關的基因，為了能進一步篩選與吞噬抗性有關之基因，本研究使用黏菌(Dictyostelium discoideum)模式進行篩選。黏菌的變形蟲(amoeba)時期與哺乳類的巨噬細胞有許多相似的特徵，其一便是具有吞噬並殺死細菌的能力。本研究以此模式篩選造成肝膿瘍且對於吞噬作用具有抗性的克雷伯氏肺炎桿菌(NTUH-K2044)之突變株庫，找尋與抵抗吞噬作用相關的致病基因，約2500株突變株之中有29個突變株可產生溶菌圈。分析跳躍子嵌入之位置，共有六個基因，其中三個為負責莢膜合成之基因，另外三個分別為KP2125(肉鹼carnitine代謝相關)，clpX(蛋白酶ATPase分子)以及wzm基因(脂多醣O抗原之運輸蛋白)，再一次將基因剔除以及將此基因補回，並確認吞噬作用的結果發現只有有△clpX 以及△wzm 基因剔除株對黏菌吞噬失去抗性，而基因補回株也恢復其抗性。此外，由健康人周邊血液分離的噬中性白血球與菌株作用證實兩突變株的確較易被人類的噬中性白血球吞噬清除。之後藉由動物實驗發現兩突變株感染之小鼠相較於野生株感染的小鼠存活率高，顯示這兩個基因確實影響克雷伯氏肺炎桿菌的致病力。此外，同時發現與clpX屬於同一個操縱基因組(operon)的clpP 基因對於抗吞噬作用也是重要的。利用基因微陣列進一步分析△clpX 基因剔除株中基因表現情形發現其莢膜合成基因表現量相較於野生株下降了3 倍，但莢膜合成量減少並不是此突變株抵抗吞噬作用能力下降的唯一原因。因此，本研究利用黏菌模式發現對於克雷伯氏肺炎桿菌抗吞噬與致病重要的基因，也證實黏菌模式可用於具有厚層莢膜之細菌以進行與吞噬抗性相關基因的篩選。

Phagocytosis is the first line of host defense against invading microorganisms, therefore, it plays an important role in host antibacterial response. On the other hand, some bacterial genes responsible for attacking phagocytosis may be essential for their pathogenicity. Klebsiella pneumoniae causing pyogenic liver abscess is an important emerging infectious disease in Taiwan. Resistance to innate immune response —serum killing and phagocytosis are known to be critical virulence factors in K. pneumoniae. Using transposon mutagenesis and mucoviscosity screening, we had identified a virulent gene, magA, related to phagocytic resistance. This gene was further proved to be associated with synthesis of K1 capsule. Because the limitation of screening by mucoviscosity, we use a Dictyostelium model to identify genes associated with phagocytic resistance. Since the amoeba form of Dictyostelium discoideum and mammalian macrophage share many traits such as the ability to phagocytose and kill bacteria, in this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, 6 loci were identified; three were capsular synthesis genes. Among the other three, one was related to carnitine metabolism (KP2125), one encoded a subunit of protease (clpX), and the other encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only △clpX and △wzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of △clpX was examined by microarray and revealed a 3-fold lower level of expression of capsular synthesis genes. However, decrease of capsule synthesis was not the only reason for impaired phagocytosis resistance. Therefore, we have identified genes involved in resistance to phagocytosis and virulence in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily-encapsulated bacteria.