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

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.