探討Lpp在尿道致病性奇異變形桿菌中扮演之角色

The role of Lpp in Uropathogenic Proteus mirabilis

郭玥婷(Yueh-Ting Kuo)

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/NTU202102481

摘要

奇異變形桿菌(Proteus mirabilis)為一臨床重要尿道致病菌，尤其常見感染長期使用尿導管的病人。其為革蘭氏陰性菌，具有外膜、肽聚醣、內膜的三層包膜(envelope)結構。Lpp存在於外膜蛋白且以共價鍵連接肽聚醣，能夠維持細胞形狀以及膜間腔(periplasm)的距離。於陰性菌，Lpp是含量最豐富的外膜蛋白之一。目前已有眾多研究表明Lpp參與細菌對於外膜壓力的抵抗機制，且影響外膜的通透性。本篇研究欲探討Lpp對於Proteus mirabilis的重要性以及對毒力因子的影響。首先建構並確認lpp突變株，並分析其外膜組成，再分析lpp突變株對於Proteus mirabilis的表現型，發現lpp突變株的表面移行能力下降，且對於氧化壓力的抵抗能力，及巨噬細胞內存活能力皆不如野生株。以最小抑制濃度實驗分析lpp突變株，則發現其對於某些藥物以及SDS的感受性上升。另外也發現，lpp突變株的外膜通透性相較於野生株高。lpp突變株的細胞入侵能力及細胞毒性相較於野生株顯著下降，所引起的促發炎細胞因子表現也相較野生株弱。而以小鼠模型分析其膀胱和腎臟的定殖能力，發現lpp缺失導致P. mirabilis膀胱定殖能力顯著降低。其後探討了lpp突變株的基因調控，發現lpp缺失引起調控外膜壓力的rpoE, cpxP表現量提升。在缺失hfq及rpoE的情況下，lpp表現量則下降，表明hfq及rpoE參與Lpp的調控路徑。由上述可得知，Lpp在Proteus mirabilis中於外膜組成、壓力抵禦機制及泌尿道感染上扮演重要角色，具備作為抗感染藥物標的的潛力。

關鍵字

奇異變形桿菌 ； lpp ； 外膜蛋白 ； 壓力抵抗 ； 毒力因子

並列摘要

Proteus mirabilis is an important pathogen of urinary tract infection clinically, espesically in patients with long-term catheterization. The structure of the Gram-negative bacterium envelope has three layers: outermembrane, peptidoglycan and innermembrane. As an outermembrane protein, Lpp covalently binds to peptidoglycan, and is able to stabilize the cell shape and periplasme space. In gram-negative bacteria, Lpp is one of the most abundant outer membrane protein. Recently, many studies suggest Lpp participates in the mechanism of environmental stress resistance, and affects the permeability of outer membrane. We investigate the importance of Lpp to P. mirabilis and whether it contributes to virulence factors. First of all, we constructed and confirmed lpp mutant and analyzed the composition of the outer membrane and phenotype. We found swarming motility, the resistance to oxidative stress and survival in macrophage decreased in lpp mutant. We further found the susceptibility of specific antibiotis and SDS and the membrane permeability increased in lpp mutant. In addition, the influence of lpp knockout had also shown by significantly lower cell invasion ability, cyototoxicity, and cytokine production in acute inflammation. In mouse model, the colonization of bladder declined with lpp mutant infection. We next investigate gene regulation in lpp mutant. The expression level of rpoE and cpxP, which are related to envelope stress reponses, elevated as lpp gene was deleted. In addition, lpp expression level reduced in hfq and rpoE knockout mutant, which suggests hfq and rpoE participate in the lpp regulation pathway. Overall, Lpp plays an important role in swarming, stress resistance and urinary tract infection in P. mirabilis, which inditates the potential for Lpp as a target of anti-infectives against P. mirabilis.

並列關鍵字

Proteus mirabilis ； lpp ； outer membrane protein ； stress resistance ； virulence factor

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