雙組成系統之反應調節子-NtrC於尿道致病性奇異變形桿菌中扮演之角色

The role of NtrC, a two-component system response regulator, in uropathogenic Proteus mirabilis

陳昱璇(Yu-Hsuan 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(2022年)

https://doi.org/10.6342/NTU202204052

摘要

奇異變形桿菌(Proteus mirabilis)是造成伺機性尿道感染之常見致病菌。NtrBC two-component system為氮源調控之主要系統，而其亦是影響許多細菌毒力之重要調節子，根據先前實驗室研究，氮源調節相關之sigma factor-RpoN的缺乏使P. mirabilis表面移行能力(swarming)下降，NtrC為RpoN enhancer binding protein之一，且諸多文獻指出ntrC的缺失亦影響其他細菌之運動性與諸多毒力因子，因此我們擬分析ntrC於P. mirabilis中所扮演之角色，並探討其調控機制。
首先經動物實驗結果顯示，ntrC突變株於小鼠之膀胱與腎臟定殖能力皆較野生株差，說明了ntrC於P. mirabilis致病力中扮演著重要的角色，我們接著分析ntrC的缺失所影響之毒力因子。結果可知ntrC突變株相較於野生株，其運動性、壓力抗性與胞外多醣體分泌能力皆下降，而細胞貼附能力、促進細胞激素之分泌則較野生株上升。此後經reporter assay與RT-qPCR進行基因分析可知， NtrC透過正調控flhDC促進鞭毛蛋白生成，讓細菌具備較好的運動性，而當其分化成swarmer cell所分泌的溶血素，亦使之具備較佳溶血能力。當P. mirabilis面臨壓力時，NtrC會上調節spoT，因此由抗壓相關表現型可觀察到ntrC突變株於酸性、氧化壓力環境中與巨噬細胞試驗之生存率較野生株下降；NtrC亦會正調控cpsF來促使胞外多醣體之分泌，增加壓力抗性與作為表面移行時所需之潤滑劑。檢測其纖毛相關基因顯示NtrC負調控pmpA，因此由細胞貼附試驗結果可知ntrC突變株貼附能力較野生株上升。最後則觀察到ntrC突變株較易促使細胞激素分泌。綜合上述可知，ntrC於P. mirabilis致病力中扮演著重要的角色。
NtrC可獨立轉錄亦可與glnA共用promoter一起轉錄，且NtrC具自我調控機制，會抑制ntrBC與glnA表現量。最後由reporter assay結果顯示，硫酸銨濃度逐漸提高的情況下，NtrC逐漸喪失對glnK-amtB promoter之促進能力，表示NtrC會在低濃度硫酸銨時受活化，而當處於硫酸銨充足的情況下，NtrC活性則下降。為探討NtrB是否只透過磷酸化NtrC來調控表現型，我們將ntrB基因剔除，分析ntrB突變株之表現型，並比較結果是否與ntrC突變株一致。結果顯示，ntrB突變株與ntrC突變株表現型趨勢相同為表面移行能力、泳動能力、酸性與氧化壓力抗性與生物膜生成能力，相異的部分為ntrB突變株之尿素酶活性較野生株顯著下降，因此我們推測NtrB會cross-regulate其他response regulator。
總結以上實驗結果，NtrC促進小鼠腎臟與膀胱中之定殖，正向調控flhDC、spoT與cpsF來分別提升表面移行能力、抗壓能力與增加胞外多醣體生成。而ntrC突變株PmpA纖毛表現量增加也較易促使細胞激素之分泌。由上述可知NtrC於P. mirabilis之致病力中扮演重要的角色。

關鍵字

尿道致病菌 ； 奇異變形桿菌 ； NtrC ； 氮源調節子 ； 毒力因子

並列摘要

Proteus mirabilis is an opportunistic pathogen commonly causing urinary tract infection. NtrBC, a two-component system mainly serves to regulate nitrogen utilization, also contributing to virulence factors expression such as motility in various bacterial species. Our previous studies showed lack of sigma factor-RpoN leads to a decreased swarming ability. In view that NtrC is one of the enhancer binding proteins on RpoN, we wondered if ntrC is involved in the RpoN-mediated phenotypes. Therefore, the purpose of this study is to disclose the role of ntrC in P. mirabilis and investigate the underlying mechanisms.
First, we confirmed the ntrC mutation had no effect on growth and performed the mouse colonization experiment. The ntrC mutant showed a significant reduction in both kidney and bladder colonization compared with the wild-type, indicating the importance of ntrC in pathogenicity. We next analyzed the impact of ntrC deficiency on virulence factor expression. The results showed that ntrC mutation had no effect on urease activity and biofilm formation ability but a significant decrease in swarming ability, swimming ability, flagellin level, stress-resistance and exopolysaccharide (EPS) formation. The ntrC mutant exhibited higher adhesion ability and induced higher cytokine production than the wild-type strain. The reporter assay revealed that NtrC positively regulated flhDC promoter activity. The ntrC mutant had lower spoT (a gene coping with stress environment) expression by the reporter assay and RT-qPCR. Accordingly, we observed that ntrC mutant had a lower survival rate on exposure to low pH or menadione and in macrophages than the wild-type. NtrC also upregulated cpsF expression to promote production of EPS which protects bacteria from stresses and also increases medium surface fluidity to facilitate swarming migration. We further constructed ntrB mutant and analyzed related phenotypes of ntrB mutant. Data showed that swarming ability, stress resistance and biofilm formation were similar to the ntrC mutant. Wherase, the urease activity of ntrB mutant was different from ntrC mutant. Thus, NtrB may cross-regulate other response regulators. Moreover, NtrC positively regulates expression of PmpA pili. NtrBC can be transcribed independently or co-transcribed with glnA and acts as a repressor of ntrBC and glnAp1 promoter. We identified that low concentration of ammonium acts as a signal for the NtrC-transduction pathway by the reporter assay using NtrC-regulated glnK promoter.
Together, NtrC positively regulates flhDC, spoT and cpsF to promote swarming ability, stress-resistance and EPS production, respectively, and inhibits cytokine production, contributing to the mouse colonization. In conclusion, ntrC plays an important role in the pathogeneicity of P. mirabilis.

並列關鍵字

uropathogen ； Proteus mirabilis ； NtrC ； nitrogen regulator ； virulence factor

參考文獻

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Copyright © 2022, StatPearls Publishing LLC.: Treasure Island (FL).

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