克雷白氏肺炎桿菌CG43磷酸二酯酶YjcC與二級訊息分子c-di-GMP在壓力反應調控之功能性研究

Abstract

克雷白氏肺炎桿菌是一株伺機性的革蘭氏陰性病原菌，可造成肺炎、原發性肝膿瘍、尿道和化膿性感染、及敗血症等廣泛疾病。我們實驗室先前利用小鼠活體表現技術（in vivo expression technology），由克雷白氏肺炎桿菌CG43基因庫中分離出只在小鼠體內表現、影響其毒性且會受巴拉刮（1,1'-二甲基-4,4'-聯吡啶氯化物）誘導表現的yjcC基因，分析此基因序列顯示此具534個殘基的蛋白質N端具有2個穿膜的二級結構和可能接收訊息的CSS功能區，其C端具有磷酸二酯酶（phosphodiesterase; PDE）活性的功能區。本論文的第一部份，我們證實YjcC可受巴拉刮和過氧化氫誘導表現；剔除yjcC基因除了使CG43抗氧化壓力的能力減低外，也降低其莢膜多醣的合成及對小鼠的毒性；而yjcC基因缺損株中的活性氧自由基、氧化傷害化、第三型線毛單位蛋白MrkA表現及生物膜活性相對增加；同時，經純化的YjcC磷酸二酯酶功能區重組蛋白被證實具有PDE活性；進一步，在CG43增加YjcC表現使c-di-GMP濃度降低後的mRNA定序結果顯示有34與29個基因分別因而提高和降低其表現量，而其中包括與氧化壓力相關的基因。這些結果顯示YjcC在氧化壓力反應中扮演正向調控的角色（詳述於第二章）。 細菌的二級訊息分子cyclic di-GMP（c-di-GMP）由雙鳥苷酸環化酶（diguanylate cyclase; DGC）環化合成，被磷酸二酯酶所分解，因此，c-di-GMP於細胞內的濃度由此兩群酵素活性來調控。最近許多的報導顯示c-di-GMP訊號 IV 傳遞系統主導許多基因的表現與生理功能的調控。為了進一步探討克雷白氏肺炎桿菌中c-di-GMP濃度是否參與調控細菌對抗各種壓力的反應，我們將具有DGC活性的質體pRK415-ydeH轉殖至K. pneumoniae CG43S3提高c-di-GMP的濃度後，再以轉錄體RNA定序與定量分析受影響的基因，結果顯示除了已被證實受c-di-GMP濃度正向調控的基因mrkABCDF和mrkHI表現量明顯升高外，熱休克反應及其它壓力反應相關的基因ibpA、clpB、dnaK、grxA和dinI也大量表現；而比較K. pneumoniae CG43S3[pRK415]和CG43S3[pRK415-ydeH]發現細胞內的c-di-GMP濃度上升使細菌抵抗50℃的熱休克反應的能力增加，相對的卻降低細菌對抗氧化及酸壓力，此結果暗示細菌可藉改變c-di-GMP濃度來調控抵抗惡劣環境的反應（詳述於第三章）。 K. pneumoniae CG43基因體已於今年初定序完成，序列分析結果顯示至少有25個與c-di-GMP濃度調節相關的基因，釐清這些基因的表現調控和功能性，將有助於建立c-di-GMP主導調控的基因網路，也將有利於了解c-di-GMP在細菌對抗壓力反應的調控機制。另外，最近研究證實c-di-GMP可與STING蛋白結合而增強人類干擾素的表現，又因其增強免疫反應的特性，可提供作為疫苗佐劑；未來，如何應用c-di-GMP配合抗生素的使用來降低細菌的感染將是一個值得深入探討的課題。

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that causes a wide range of infections, including pneumonia, urinary tract, purulent infections, primary liver abscess and septicemia. We have previously identified from K. pneumoniae CG43, using in vivo expression technology (IVET), yjcC gene which was shown to be inducible by paraquat and affect its virulence to mouse. Sequence analysis of YjcC shows a signal peptide followed by 2 transmembrane domains and a CSS motif at the N-terminal region, whereas the C-terminal contains a conserved EAL domain of the PDE enzyme. For the first part of the thesis, we have demonstrated that yjcC is induced expression by paraquat and H2O2. The yjcC deletion reduced the bacterial oxidative stress resistant activity, capsular polysaccharide production, and virulence to mouse. In addition, the yjcC deletion mutant exhibited increased production of reactive oxygen species, oxidative damage, type 3 fimbriae MrkA pilin, and biofilm. The recombinant protein containing the YjcC-EAL domain was demonstrated to exhibit phosphodiesterase (PDE) activity. Moreover, transcriptome analysis via RNAseq of CG43S3[pRK415-yjcC] compared to the CG43[pRK415] gene expression revealed 34 upregulated and 29 downregulated genes, which include stress related genes. The results suggest that yjcC plays a positive role in the anti-oxidative stress regulation in K. pneumoniae CG43S3 VI (Detailed description in Chapter 2). In bacteria, the second messenger c-di-GMP is regulated by diguanylate cyclase (DGC) enzymes and phosphodiesterases (PDEs) that catalyze synthesis and hydrolysis of this molecule, respectively. Many recent reports show that the c-di-GMP-mediated signal transduction system is a major regulator for many gene expression and physiological response. In order to investigate if c-di-GMP level is involved in the stress response regulation in K. pneumoniae, the DGC expression plasmid pRK415-ydeH was used to transform K. pneumoniae CG43S3 to elevate the intracellular c-di-GMP level. Subsequently, transcriptome analysis via RNAseq was employed and then qRT-PCR analysis used to confirm the affected genes. The results showed that, in addition to the reported c-di-GMP upregulated genes mrkABCDF and mrkHI, the heat shock response and other stress response genes including ibpA, clpB, dnaK, grxA, and dinI are also increasingly expressed. Compared to CG43S3[pRK415], CG43S3[pRK415-ydeH] had increased the heat shock (50℃) resistant activity, but had reduced the bacterial resistance to oxidative and acid stress by the increase of the c-di-GMP levels. The results imply that bacteria could resist to the harsh environment by modulating the intracellular c-di-GMP levels. Analysis of the recently resolved genome sequence of K. pneumonia CG43 revealed 25 genes coding for GGDEF- or EAL-domain proteins that are responsible VII for the intracellular c-di-GMP level modulation. To clarify how these gene expression are regulated and what are their functional roles in the bacteria should help to establish a c-di-GMP-dependent gene network. These data should also provide much more insights of the c-di-GMP role in the stress response regulation. Besides, several recent reports have demonstrated that c-di-GMP is able to bind STING protein, the stimulator of interferon gene, thereby enhances the immune response. It is proposed to be used as vaccine adjuvant because of the potent immune modulator property. In the future, the issue of how to apply c-di-GMP together with antibiotic treatment to decrease bacterial infections deserves an in depth investigation.