從突變基因庫找出克雷伯氏肺炎桿菌參與腸道細胞黏附作用相關的基因

Abstract

克雷伯氏肺炎桿菌(Klebsiella pneumoniae)為一種伺機性感染的病原菌，常引起肺炎、敗血症、泌尿系統發炎及社區性肝膿瘍等感染。細菌附著於宿主細胞通常是達成感染的第一步驟，因此黏附因子(adhesin)可能在細菌貼附到細胞的過程中扮演重要角色。克雷伯氏肺炎桿菌為腸道中的正常菌叢，最近我們的研究也顯示在腸道中克雷伯氏肺炎桿菌會穿過(translocation)腸道上皮而造成感染，因此我們想找尋克雷伯氏肺炎桿菌與腸道上皮細胞貼附相關的基因。利用跳躍子(transposon)建構了克雷伯氏肺炎桿菌血清型K2菌株Ca0437之突變株庫(mutant library)，此突變株庫包含4600株的突變株，以Caco-2細胞作黏附試驗(adherence assay)，一共篩選了2450株突變株，發現其中有9株突變菌株其黏附能力有明顯的下降，以NCBI-BLAST作基因功能比對分析，這些被跳躍子破壞的基因可分為含有跨膜區域(transmembrane domain)的基因、調控(regulator)基因、酵素(enzyme)與其它(others)。利用無標誌基因剔除法(unmarked gene deletion)方式分別建構sapA、dgc與hns基因剔除菌株，驗證這些基因剔除菌株之細胞黏附能力下降，且細菌生長試驗證實非因生長差異所致。也建構了相對之染色體互補(chromosome complementation)菌株，發現dgc與hns基因染色體互補菌株的細胞黏附能力有補回，而sapA基因剔除菌株則是利用質體補回操縱子sapABC使得其細胞黏附能力回復。此外，hns為一個上游之調控基因，進一步利用基因微陣列分析其調控克雷伯氏肺炎桿菌之基因轉錄，發現可影響第一型線毛的相關基因表現。分別以胃內感染和腹膜注射感染小鼠的方式評估sapA、dgc與hns基因剔除株對於致病力(virulence)的影響，發現在胃內感染的情況下，hns基因剔除株的致病力較野生株有明顯下降，其餘的基因剔除菌株則與野生株並無顯著差異，因此未來會對基因剔除株的致病力進行實驗來作進一步探討。

Klebsiella pneumoniae is an opportunistic pathogen associated with pneumonia, septicemia, urinary tract infection, pyogenic liver abscess and other infections. Bacterial adherence to host cells is usually the first step in pathogenesis of infections, and therefore adhesins play an important role in the process. K. pneumoniae is the normal intestinal flora, and recent studies showed translocation of K. pneumoniae through the intestinal epithelial cells leading to dissemination of the infection into the bloodstream. Hence, we wanted to explore the genes responsible for adhesion to intestinal epithelial cells in K. pneumonia. We constructed the transposon mutant library of K. pneumonia serotype K2 strain Ca0437, and the library included 4600 mutants. The mutant library was screened by adherence assay with Caco-2 cells. A total of 2450 mutants were screened and nine mutants decreased in the ability of adhesion to cells. Through NCBI-BLAST for gene function analysis, the gene inserted by transponsons can be divided into the genes with transmembrane domain, regulatory genes, enzymes and others. Unmarked deletion and complementation of sapA, dgc and hns gene demonstrated that they were responsible for adhesion to cells, instead of the differences of bacterial growth rate. In order to study the role of gene regulation in hns gene, we compared the RNA expression profiles of Ca0437Δhns with those of Ca0437 wild-type by microarray. The two clones which expressions were down-regulated in Ca0437Δhns contained genes in association with type 1 fimbriae. In vivo experiments showed that intragastric infection of Δhns mutant had significant differences with the wild-type strain. And we will study the virulence of the other deletion mutants in the future.