克雷伯氏肺炎桿菌之血清誘發鐵離子攝取系統和 TonB 蛋白質對其致病力的影響

Abstract

本論文利用基因微陣列方法來研究克雷伯氏肺炎桿菌於健康人血清（human serum, HS）中之血清誘發基因的表現，發現到三組受血清誘發表現的鐵離子攝取系統與臨床上造成社區型細菌性肝膿瘍之克雷伯氏肺炎桿菌具有關聯性：Yersinia high-pathogenicity island（Yersinia HPI）、iucABCDiutA 和 iroA 基因群組，利用交互飼養試驗（cross-feeding assay），已證實這 3 段基因群組之鐵離子攝取系統的功能。只有注射 irp2 iuc iroA triple mutant 此多重基因剔除菌株於鼷鼠動物模式會降低此菌的致病力。由克雷伯氏肺炎桿菌 NTUH-K2044 菌株之全基因體序列分析，共發現 10 組鐵離子攝取系統，其中有 7 組皆為 TonB-依賴型之鐵離子攝取系統，包括 Yersinia HPI 、 iucABCDiutA 和iroA 基因群組。於鼷鼠動物模式中， tonB 基因剔除菌株大幅度地減弱此菌的致病力。以腹膜腔注射 tonB 基因剔除菌株來免疫鼷鼠，結果顯示鼷鼠血清中之莢膜抗體陽轉且對於後續原生菌株之感染具保護力。總結，鐵離子攝取系統是造成社區型細菌性肝膿瘍之克雷伯氏肺炎桿菌主要的血清誘發基因，雖然在 NTUH-K2044 菌株中存在很多組的鐵離子攝取系統，只有 irp2 iuc iroA 之多重基因剔除菌株才能減弱此菌的致病力，活性減毒 tonB 基因剔除菌株具有作為疫苗株之潛力，可誘發鼷鼠產生具顯著性之對抗原生菌株的保護性免疫反應。

Klebsiella pneumoniae was stimulated by human serum and gene expression was analyzed by microarray. Three putative iron acquisition systems, Yersinia high-pathogenicity island（Yersinia HPI）, iucABCDiutA and iroA(iroNDCB), that increased in expression and predominate in PLA-associated K. pneumoniae strains were identified. By siderophore uptake assays, these three systems were confirmed to be siderophore-dependent iron acquisition systems. Only the irp2 iuc iroA triple mutant showed decreased virulence in mice. Full genome analysis of K. pneumoniae NTUH-K2044 identified 10 putative iron uptake systems. Seven of them were TonB-dependent, including Yersinia HPI, iucABCDiutA and iroA. A tonB deletion mutant was demonstrated profound attenuation of virulence. Immunization with the tonB mutant showed sero-conversion of extracellular polysaccharides antibody and protective efficacy against subsequent exposure to the parental strain. Taken together, iron uptake systems were the highly up-regulated genes in K. pneumoniae in response to sera. Although there are multiple iron transporter systems in NTUH-K2044, a mutation of all three loci, irp2, iuc, and iroA, is necessary to decrease virulence. The tonB mutant is a potential vaccine candidate because it can induce significant protective immune response against wild-type strain challenge.