HP1248為3端至5端核醣核酸外水解酶之基因並調控幽門螺旋桿菌運動性與細胞凋亡引發因子等致病相關基因之表現

The 3'-to-5' exoribonuclease (Encoded by HP1248) of Helicobacter pylori regulates motility and apoptosis-inducing genes

曹銘陽(Ming-Yang Tsao)

指導教授 ： 王錦堂

國立臺灣大學/醫學院/微生物學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02427

摘要

幽門螺旋桿菌 (Helicobacter pylori)，人類消化道相當重要的病原菌，有著許多的毒性因子參與致病過程，但是這些毒性因子的調控機制卻還未被全部了解。在此研究中，我們利用一幽門螺旋桿菌的表現基因庫，經由chlorhexidine的篩選，選殖到了 HP1248可能是一個抗chlorhexidine的相關基因，其序列相似於大腸桿菌的vacB序列；但進一步研究卻顯示出此結果可能是過度表現所造成的人工現象或間接影響。在之前研究，vacB與志賀氏菌屬(Shigella)和腸侵入型大腸桿菌(enteroinvasive E. coli.)的毒性表現有密切相關。大腸桿菌的vacB 表現出的蛋白質是RNase R。RNase R參與在後轉錄(post-transcriptional)層級中mRNA的穩定性調控。藉由幽門螺旋桿菌HP1248剔除突變菌株全面性的轉錄層級微陣列分析，我們找到六個致病相關基因在後轉錄層級中被HP1248所抑制表現，包括了運動性相關基因：HP1192和flaB，趨化性相關基因：cheY，和細胞凋亡引發因子相關基因：HP0175，cagA和 gtt。在本篇研究中，在大腸桿菌表現的重組HP1248 蛋白質可表現出3端至5端核醣核酸外水解酶活性。在幽門螺旋桿菌HP1248剔除突變菌株的運動能力和引發宿主細胞的細胞凋亡能力也都有顯著增加。我們的研究也顯示HP1192和幽門螺旋桿菌的運動性確實有相關，而且很可能是經由HP1248的調控。更進一步，我們亦研究並推測出被HP1248所特定調控的這些致病相關基因的可能的機制。除此之外，HP1248 mRNA的表現量會隨著不同的環境因子而有明顯的改變，包括pH值和溫度。因此，幽門螺旋桿菌的HP1248 似乎在對外在環境的感受扮演相當重要的角色，並且進一步來調控細菌的運動性與引發宿主細胞凋亡的相關因子...等致病相關的基因表現。

關鍵字

幽門螺旋桿菌 ； 核醣核酸外水解酶致病基因

並列摘要

The human gastric pathogen Helicobacter pylori has many virulence factors involved in pathogenesis, but the mechanisms regulating these virulence factors are not yet fully understood. In this study, we cloned HP1248 during a screen for chlorhexidine-resistant clones in an expression library of H. pylori. HP1248, which is similar in sequence to Escherichia coli vacB, was previously shown to be associated with the expression of virulence in Shigella and enteroinvasive E. coli. E. coli vacB encodes RNase R. RNase R is involved in the post-transcriptional regulation of mRNA stability. By global transcriptional microarray profiling of an H. pylori HP1248-deletion mutant, we defined six virulence related genes which were post-transcriptionally down-regulated by HP1248, including the motility related genes HP1192 and flaB, the chemotaxis related gene cheY, and the apoptosis-inducing genes HP0175, cagA, and gtt. In this study, recombinant HP1248 protein expressed in E. coli showed 3'-to-5' exoribonuclease activity. Motility and apoptosis-induction were increased in the H. pylori HP1248-deletion mutant. We also showed that HP1192 is associated with H. pylori motility, possibly through HP1248 regulation. Further, we suggested and studied the possible mechanisms of this specific regulation of virulent genes by HP1248. In addition, the expression level of HP1248 mRNA changes dramatically in response to a variety of altered environmental conditions, including pH and temperature. Hence, HP1248 in H. pylori seems to play a role in environmental sensing and in regulation of virulent phenotypes, such as motility and host-apoptosis induction.

並列關鍵字

Helicobacter pylori ； exoribonuclease ； HP1248

參考文獻

1. Alamuri, P., and R. J. Maier. 2004. Methionine sulphoxide reductase is an important antioxidant enzyme in the gastric pathogen Helicobacter pylori. Mol. Microbiol. 53:1397-1406.

2. Amblar, M., and C. M. Arraiano. 2005. A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding. FEBS J. 272:363-374.

3. Andrade, J. M., F. Cairrao, and C. M. Arraiano. 2006. RNase R affects gene expression in stationary phase: regulation of ompA. Mol. Microbiol. 60:219-228.

4. Ang, S., C. Z. Lee, K. Peck, M. Sindici, U. Matrubutham, M. A. Gleeson, and J. T. Wang. 2001. Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray. Infect. Immun. 69:1679-1686.

5. Basak, C., S. K. Pathak, A. Bhattacharyya, S. Pathak, J. Basu, and M. Kundu. 2005. The secreted peptidyl prolyl cis,trans-isomerase HP0175 of Helicobacter pylori induces apoptosis of gastric epithelial cells in a TLR4- and apoptosis signal-regulating kinase 1-dependent manner. J. Immunol. 174:5672-5680.