探討尿道致病性奇異變形桿菌之抗銅機制-銅結合蛋白質CueR抗銅及調控毒力因子之研究

Characterization of copper resistance in uropathogenic Proteus mirabilis-the role of copper regulator CueR in copper resistance and virulence

王璐(Lu Wang)

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(2021年)

https://doi.org/10.6342/NTU202102493

摘要

奇異變形桿菌(P. mirabilis)為人體一重要的尿道致病菌，其常引發尿導管相關的尿道感染。當尿道感染發生時人體為抵禦病菌的入侵會提升尿液中的銅濃度，其中P. mirabilis所引發的UTI可使尿液中銅濃度上升至約0.5 μM，同時清除外來致病菌的巨噬細胞中銅濃度會提升至約500 μM來加强對致病菌的毒殺作用。過量的銅會對菌體造成傷害，故細菌會啓動抗銅機制來維持菌體内銅濃度之恆定。目前對P. mirabilis的抗銅機制所知甚少，故本篇欲探究P. mirabilis的抗銅機制。
首先利用跳躍子突變法從約15000株突變株中篩選得到5株對銅感受性提高之突變株，經過基因分析後得到cueR突變株及copA突變株；同時以blast方式來尋找P. mirabilis中存在的抗銅基因，並結合實驗室之前transcriptome之結果推測出Cue系統於P. mirabilis的抗銅機制中扮演著重要作用。於是利用reporter assay及qPCR確認了P. mirabilis之CueR可正向調控抗銅相關基因cueO、 copA及copG之表達，而AAS之結果亦印證CueR可幫助菌體維持菌體内之銅濃度恆定。
接著探究CueR在0.5 μM及500 μM這兩個已知的生理銅濃度下對菌體毒力因子之影響，本篇結果表明無銅或0.5 μM銅離子環境并不影響cueR突變株生長狀況及細胞外毒力因子之表達，但於500 μM 銅離子環境下，cueR突變株相較於野生株之生長狀況、尿素酶活性、生物膜形成能力、表面移行能力及泳動能力皆顯著下降，由reporter及qPCR之分析可知cueR突變株因鞭毛形成相關基因flhDC的轉錄及fimbriae中與感染力相關之基因mrpA、pmfA、fim14、pmpA之表現量顯著下降，進而造成其運動性及細胞黏附能力下降。而在預處理銅後其抗氧化能力及巨噬細胞内存活率顯著上升可能與scsA表現量增加有關，但在未預先處理銅時cueR突變株之抗氧化能力及巨噬細胞内存活率則顯著低於野生株，同時小鼠實驗亦證實cueR突變株於小鼠膀胱及腎臟中的定植能力均顯著下降。
綜上可知於銅環境下CueR不僅可正向調節cueO、copA及copG之基因表達來維持菌體内銅濃度水平，亦可影響尿素酶活性、運動力、生物膜形成能力、上皮細胞黏附能力、巨噬細胞内存活能力及其定植能力等多種毒力因子。因此CueR在P. mirabilis之抵抗銅壓力及維持毒力因子中均扮演著重要角色。

關鍵字

尿道致病菌 ； 奇異變形桿菌 ； 銅 ； 銅調控子CueR ； 毒力因子

並列摘要

Proteus mirabilis is an important pathogen of the urinary tract, commonly causing catheter-associated urinary tract infections (CAUTI). Urine copper concentration will increase to defend against bacterial intrusion upon urinary tract infection. The copper concentration of human urine and the phagosome will increase to about 0.5 μM and 500 μM, respectively to protect from bacteria infection. The presence of excessive copper is detrimental to the bacteria, so the bacteria armored themself with a series of anti-copper mechanisms to reduce the toxicity of copper and maintain the copper homeostasis.
The copper resistance mechanisms of P. mirabilis are not clear. To investigate the resistance mechanisms, we isolated copper-sensitive mutants by transposon mutagenesis. After screening about 15000 mutants, 5 mutants with high sensitivity to copper were isolated and the cueR mutant and copA mutant were obtained after further analysis. At the same time, we used Blast and the transcriptome to search for the copper resistance genes in P. mirabilis. The results show that the Cue system could be the major copper resistance player in P. mirabilis. By the reporter assay and qPCR, we found that the CueR can positively regulate the expression of copper resistance genes including cueO, copA and copG. The results of Atomic Absorption spectrometry (AAS) also confirmed that CueR plays a role in maintaining the copper homeostasis.
We next explored the effect of CueR on the virulence factors of P. mirabilis in the presence of copper at concentration of 0.5 μM and 500 μM. The results show that the cueR mutation had no effect on the growth and virulence factors at 0.5 μM copper. While at 500 μM Cu, the growth, urease activity, biofilm formation ability, swarming and swimming ability of the cueR mutant were significantly reduced compared with the wild type. The expression of flagellar master regulator FlhDC and the infectivity-related fimbrial genes, mrpA, pmfA, fim14, and pmpA of cueR mutant were significantly decreased by qPCR analysis. Accordingly, the motility and cell adhesion ability were decreased. In addition, copper pretreatment significantly increased the survival under oxidative stress and intra-macrophage, which may be related to the increased expression of scsA. However, without copper pretreatment, the survival of the cueR mutant under oxidative stress and intra-macrophage significantly decreased compared to the wild type. Finally, the mouse experiments showed that the colonization of the cueR mutant in the bladder and the kidney was significantly reduced relative to the wild-type.
Overall, this study shows that in the presence of copper, CueR not only maintains the copper homeostasis of P. mirabilis by positively regulating the gene expression of cueO, copA and copG, but it also affects the expression of multiple virulence factors of the bacteria, including urease activity, motility, biofilm formation, epithelial cell adhesion ability, intra-macrophage survival and mouse colonization ability. Therefore, CueR plays an important role in copper resistance and pathogenicity of P. mirabilis.

並列關鍵字

uropathogen ； Proteus mirabilis ； copper ； copper regulator CueR ； virulence factor

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