探討small RNA GlmZ在奇異變形桿菌中所扮演之角色

Investigation of the small RNA GlmZ in uropathogenic Proteus mirabilis

陳力嘉(Li-Chia Chen)

指導教授 ： 廖淑貞

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601782

摘要

奇異變形桿菌 (Proteus mirabilis) 屬腸內菌科 (Enterobacteriaceae)，兼性厭氧之革蘭氏陰性桿菌，為健康人類腸道中之正常菌叢，但於免疫功能低下之病人中容易造成伺機性感染，特別在使用尿導管的病患身上，容易造成尿道感染，感染程度嚴重則可能經輸尿管上行感染至膀胱、腎臟，造成膀胱炎、腎盂腎炎、菌血症等併發症。過去的研究中，在Escherichia coli中發現GlmY與GlmZ會調控glmS (glucosamine-6-phosphate synthase) 之表現，影響細胞壁合成。另外，對E. coli O157:H7之研究結果顯示GlmY及GlmZ會幫助細菌貼附上皮細胞。根據實驗室過去之研究，雙組成系統 (two-component system) QseE/F在P. mirabilis中主要負責兩個部分：移動性與毒性。在移動性方面，QseE/F會接收環境中的訊號，調控sRNA GlmY之表現而進一步影響其下游基因cheA表現，進而影響P. mirabilis之表面移行能力。在細胞毒性的部分，qseEGF突變株對膀胱癌細胞 (NTUB1) 之入侵能力相較於野生株大幅下降。目前，在P. mirabilis中，GlmZ之功能及其參與之調控機制尚待釐清。本研究中，透過建構glmZ突變株及glmZ過度表現菌株，觀察GlmZ影響之表現型。實驗結果顯示，GlmZ不會影響P. mirabilis之生長；glmZ突變株入侵膀胱癌細胞 (NTUB1) 及腎臟癌細胞 (A498) 的能力、在巨噬細胞中的生存能力以及氧化壓力 (H2O2) 的能力皆顯著下降；glmZ突變株之表面移行能力及抗藥性則與野生株無顯著差異；glmZ過度表現菌株之生物膜形成能力及抵抗酸性環境能力 (pH=3) 顯著上升。在基因調控方面，qseF突變株中，GlmZ之表現顯著低於野生株之GlmZ表現；在培養環境中加入尿素 (urea) 或glucosamine後，GlmZ之表現也顯著下降。我們以生物資訊軟體Target RNA2預測GlmZ可能調控的下游基因，找到兩個與細胞壁合成的相關基因──glmS及murA，以real-time PCR偵測基因表現，我們發現在glmZ突變株中，glmS及murA之mRNA表現量皆顯著下降；另外，我們也以IntaRNA預測到GlmZ會結合於rpoS mRNA，RpoS為P. mirabilis之壓力調控因子，以real-time PCR偵測rpoS基因表現，發現在glmZ突變株中，rpoS之表現量顯著下降。於此研究中，我們發現P. mirabilis之sRNA GlmZ會受到QseE/F之正向調控，提高rpoS之表現，影響P. mirabilis抵抗酸性環境及氧化壓力之能力以及在巨噬細胞中之生存能力。

關鍵字

奇異變形桿菌, 壓力抵抗

並列摘要

Proteus mirabilis is a Gram-negative, facultative anaerobic bacterium, it is a part of the normal flora of human intestinal tract, meanwhile, it is also an opportunistic pathogen which causes urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs). Bacterial small RNAs are a group of small non-coding RNAs ranging from 50 to 500 nucleotides, which are involved in the post-transcriptional control of bacteria gene expression by complementary base-pairing with their target mRNAs. In Escherichia coli, sRNA GlmY and GlmZ have been found to up-regulate the translation of glmS (glucosamine-6-phosphate synthase) mRNA. In P. mirabilis, the two-component system QseE/F have been found to regulate the small RNAs GlmY and GlmZ using reporter assay in our previous study. Small RNA GlmY has been found to regulate the swarming ability of P. mirabilis, while the functioning roles of GlmZ has yet to be found. In this study, we investigated the roles of GlmZ in P. mirabilis. The expression of GlmZ was greatly down-regulated in the qseF mutant. The amount of GlmZ was also significantly reduced under the presence of urea or glucosamine. We constructed a P. mirabilis glmZ mutant and GlmZ was substituted with a kanamycin resistant cassette. In our study, we have found that glmZ mutant has a lower survival rate relative to wild-type under treatment with oxidative stress, glmZ mutant also has a reduced survival rate in macrophages. Futhermore, glmZ-overexpression strain increases the biofilm formation and the survival rate under acidic stress relative to wild-type. To identify potential targets of GlmZ, we used bioinformatic tools to predict possible GlmZ target genes and confirmed the target gene expression with real-time PCR. We have found that the expression of rpoS, murA and glmS are down-regulated in glmZ mutant. Altogether, our study indicates that sRNA GlmZ plays a role in the stress reponse regulation of P. mirabilis through up-regulating the expression of the stress response regulator, RpoS, helping P. mirabilis to survive under oxidative and acidic stress.

並列關鍵字

Proteus mirabilis, small RNA, GlmZ, stress response

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