克雷白氏肺炎桿菌CG43全基因體解序、抗酸轉錄體分析與尿素酶基因群功能探討

Whole Genome Sequencing, Transcriptome Analysis of Acid Response, and Urease Gene Cluster Characterization of Klebsiella pneumoniae CG43

林芳瑜(Fang-Yu Lin)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00288

摘要

克雷白氏肺炎桿菌屬腸道菌科，為一伺機性病原菌，感染宿主後會導致肺炎、泌尿道感染、腦膜炎、菌血症、敗血症等。在台灣，克雷白氏肺炎桿菌是主要造成糖尿病病人肝膿瘍的病原。引發肝膿瘍之克雷白氏肺炎桿菌其莢膜血清型大多數是屬於 K1 和 K2。克雷白氏肺炎桿菌 CG43 是從台灣分離自肝膿瘍病患，毒性較強的 K2 血清型菌株。本研究中將克雷白氏肺炎桿菌 CG43 基因體完整解序，其長度為 5,166,857 bp，與克雷白氏肺炎桿菌 NTUH-K2044 和 克雷白氏肺炎桿菌 MGH 78578 相似度分別為 93 % 及 94 %。克雷白氏肺炎桿菌 CG43 有 203 個不同於 MGH 78578 與 NTUH-K2044 的基因。對於腸內菌而言，抗酸能力是非常重要的，我們藉由轉錄體定序分析克雷白氏肺炎桿菌 CG43在酸性環境時基因表現的情況，結果顯示有 4.94 % 基因被誘導，18.34 % 基因被抑制表現超過 2 倍，主要分別為伴隨蛋白及麥芽糖操縱子相關之基因。此外，在克雷白氏肺炎桿菌 CG43 中存有兩套尿素酶基因組，與大多數克雷白氏肺炎桿菌只具有一套尿素酶基因組不同。尿素酶可以將尿素分解成二氧化碳和氨，提供氮源並中和酸性環境，幫助細菌生存於酸性環境。本研究建構三種尿素酶突變株 (ΔureA1、ΔureA2 與 ΔureA1 ΔureA2)。我們發現 ΔureA1 和 ΔureA1 ΔureA2在以尿素取代氯化銨的 M9 培養基中的生長的情形稍差。以 Christensen’s Urea Agar 測定尿素酶活性，結果顯示ΔureA1 和 ΔureA1 ΔureA2 幾乎無尿素酶活性。此外，在酸性環境下，野生株和三種突變株 (ΔureA1、ΔureA2 和 ΔureA1 ΔureA2) 有相似的生長曲線。根據這些結果我們推測尿素酶對於克雷白氏肺炎桿菌 CG43 在一般培養條件下之耐酸能力並非重要因子。本研究所完成之完整的克雷白氏肺炎桿菌 CG43 基因體序列可以提供我們更多資訊以進一步探討其致病因子。

關鍵字

克雷白氏肺炎桿菌 ； 基因體定序 ； 轉錄體 ； 尿素酶

並列摘要

Klebsiella pneumoniae is an important opportunistic pathogen that causes various human diseases such as pneumonia, urinary tract infection, meningitis, bacteremia and septicemia. In Taiwan, K. pneumoniae is the predominant pathogen responsible for pyogenic liver abscess in diabetic patients and K1 and K2 serotypes account for the majority of the isolates. K. pneumoniae CG43 was originally isolated from a patient with pyogenic liver abscess in Taiwan. It is a highly virulent K2 serotype strain. In this study, the whole genome sequence of K. pneumoniae CG43 was determined and annotated. The genome is 5,166,857 bp in length. The similarity of the CG43 genome sequence with that of K. pneumoniae NTUH-K2044 and MGH 78578 are 93 % and 94 %, respectively. K. pneumoniae CG43 has 203 open reading frames distinct from K. pneumoniae NTUH-K2044 and K. pneumoniae MGH 78578. Furthermore, because the ability of acid resistance is important for Enterobacteriaceae, we also performed transcriptome analysis of K. pneumoniae CG43 gene expression profile under acidic growth conditions. The data indicate that 4.94 % genes in CG43 genome were induced, while 18.34 % genes were repressed. Most of the up-regulated genes are associated with chaperone-related function and many of the down-regulated genes belong to maltose regulon. Besides, there are two urease gene clusters in K. pneumoniae CG43, different from most of K. pneumoniae that has only one urease gene cluster. Urease catalyzes the hydrolysis of the urea into carbon dioxide and ammonia. Besides providing nitrogen sources, the reaction can neutralize acidic environments, allowing pathogenic bacteria to survive the acidic conditions. Three types of urease mutant strains (ΔureA1, ΔureA2 and ΔureA1 ΔureA2) were constructed in CG43. Growth of ΔureA1 and ΔureA1 ΔureA2 double mutant was reduced in M9 minimal medium using urea as the sole nitrogen source. The two mutant strains lack urease activity as determined by Christensen’s Urea Agar. In addition, the growth of wild type and three urease mutant strains has no difference under acidic environment. The result suggests that urease is not a major factor contributing to acid tolerance of K. pneumoniae CG43 under normal cultural condition. To sum up, the complete CG43 genome sequence will provide critical information for future analysis of the virulence factors in the bacterium.

並列關鍵字

Klebsiella pneumoniae ； genome sequence ； transcriptome ； urease

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