尿道致病性奇異變形桿菌中CpxAR調控致病因子之研究

Regulation of virulence expression by CpxAR two-component system in uropathogenic Proteus mirabilis

陳弘翰(Hong-Han 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(2020年)

https://doi.org/10.6342/NTU202001499

摘要

奇異變形桿菌為格蘭氏陰性尿道致病菌，特別是長期使用導尿管之病患身上，常造成伺機性感染。CpxAR是一個雙組成系統，CpxA接收外界訊息後，會磷酸化CpxR而調控下游基因表現，我們分析cpxA及cpxR突變株的表現型。結果發現cpxA突變株不影響其表現型，但cpxR突變株其生物膜生成能力、抵抗銅、H2O2、酸性及鹼性環境能力、在巨噬細胞的存活率及老鼠colonization皆下降。
　　我們之前發現zapABCD和生物膜生成有關，此operon encodes IgA蛋白酶 (ZapA)、type I secretion system (ZapBCD); 1-2.5 mM銅會促進 P. mirabilis生物膜生成;而銅已知會活化CpxAR。此研究我們發現1 mM銅會活化CpxR增加zapABCD表現，而ZapBCD 和exopolysaccharide及eDNA等黏附因子的分泌皆有關，故可能影響initial adhesion而影響Cpx活化。而zapD及cpxR突變株其Mrp fimbriae (mrpA)的表現量皆下降，因而影響autoaggregation及細胞黏附能力。接著分析H2O2及銅的抵抗能力，我們發現0.1 mM 銅會活化CpxR而增加scsABCD，文獻報導此operon與H2O2及銅的抵抗能力有關，我們發現scsA突變會影響H2O2抵抗能力，但scsBCD突變不影響銅的抵抗能力。H2O2會使cpxR及scsA mRNA表現量上升，並且確認0.1 mM 銅是透過CpxR活化scsA而使細菌對H2O2抵抗能力上升。另外我們發現1 mM銅會活化CpxR而增加copCD表現， CopCD參與銅的運送，文獻報導其與抗銅的能力有關。此外CpxR會調控gadBC而影響細菌的抗酸能力，10 mM glutamate或glutamine會提高細菌抗酸能力，此能力有利於尿素酶活性。我們發現在酸性環境下， GadBC可將medium去酸化，促進尿素酶活性，進而影響細菌尿結石生成。最後我們發現鹼性環境會活化Cpx而增加YqjA (離子通道蛋白) 表現，此蛋白質已被報導與抵抗鹼性能力有關，並發現在鹼性環境下，Cpx也會調控P. mirabilis特有的重要毒力因子cytotoxin Pta。　　　　
　　在本次研究中，Cpx在抵抗銅、H2O2、酸性及鹼性環境能力、生物膜生成及Pta表現都扮演重要的角色，因而能影響在巨噬細胞的存活率及老鼠colonization。

關鍵字

奇異變形桿菌 ； CpxAR雙組成系統 ； 毒力因子

並列摘要

Proteus mirabilis, a gram negative and frequent uropathogen, often casuses opportunistic infection especially in patents with long-term use of urinary catheters. CpxAR is a two-compoenet system. When CpxA responds to environmental cues, it will kinase CpxR to regulate downstream genes. We analyzed the phenotypes of cpxA and cpxR mutants. We found that cpxA mutant didn’t have any impact, but cpxR mutant had significantly lower biofilm forming ability, the resistance to copper, H2O2, acid, and alkaline, survival in macrophage and colonization in mice.
　　We have found zapABCD related to biofilm forming. ZapABCD is an operon encoding IgA protease (ZapA) and type I secretion (ZapBCD). Also, wild type had better biofilm forming ability in 1-2.5 mM copper. As is known, copper could activate CpxAR. In this study, we found CpxR could be induced in 1 mM copper to increase the expression of zapABCD related to adhesion factors such as exopolysaccharide and eDNA which might influence the activation of Cpx. In addition, the expression of mrpA in zapD and cpxR mutants was lower, which influenced the ability of autoaggregation and cell adhesion. Second, we analyzed the resistance to H2O2 and copper. We found CpxR could be induced in 0.1 mM copper to increase the expression of scsABCD. According to another study, the operon was related to the resistance to H2O2 and copper. We found scsA mutant had significantly lower the resistance to H2O2, but scsBCD mutant had no impact on the resistance to copper. The expression of cpxR and scsA could be promoted in 30 mM H2O2. We also confirmed the pathway that wild type had better the resistance to H2O2 in 0.1 mM copper was based on the activation of CpxR in 0.1 mM copper to enhance the expression of scsA. Additionally, CpxR could be induced in 1 mM copper to increase the expression of copCD involved in copper transporter and the resistance to copper. What’s more. CpxR could regulate gadBC to influence the resistance to acid. It could also improve the resistance to acid in 10 mM glutamate and glutamine, which had a great benefit to urease activity. In acid, GadBC could deacidify the medium and promote urease activity to influence urinary stone. Last, in alkaline, we found Cpx could be induced to regulate YqjA and cytotoxin Pta respectively related to the resistance to alkaline and the important virulence factor in P. mrirabilis.
　　In this study, Cpx plays an important role in the resistance to copper, H2O2, acid, and alkaline, biofilm forming ability, and Pta to influence survival in macrophage and colonization in mouse.

並列關鍵字

Proteus mirabilis ； CpxAR two-component system ； virulence factor ； zapABCD ； scsABCD ； gadBC

參考文獻

參考文獻

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