尿道致病性奇異變形桿菌中一毒素-抗毒素系統的毒素蛋白Hha調控銅感受性及致病因子表現之研究

Abstract

奇異變形桿菌(P. mirabilis)是一種重要的泌尿道病原體，主要是經由尿道或導尿管進入泌尿道，爬行進入膀胱並增生，接著再沿著輸尿管上行至腎臟，造成腎臟感染。而銅是所有生命皆必需的微量元素，但過量也會對生物體造成毒性。細菌引起的泌尿道感染(urinary tract infection, UTI)會讓尿液中銅離子的濃度顯著上升，包括以P. mirabilis引發的UTI。銅在巨噬細胞亦會發揮殺菌作用。 爲了探究P. mirabilis銅感受性的機制，本篇論文首先利用跳躍子突變法(transposon mutagenesis)的方式，篩選出對3.25 mM銅濃度感受性增加的P. mirabilis突變株(Cu-susceptible突變株)，經過基因分析後得到hha突變株。本篇研究之Hha 蛋白在Escherichia coli及Salmonella中作為毒素，與antitoxin TomB構成Toxin-Antitoxin系統。在E. coli及Salmonella中有研究表明，Hha 控制致病因子基因的表達。我們的研究結果表明，Hha過表達對P. mirabilis菌體有毒性，TomB的表達則可保護菌體。hha 缺失會影響細胞對銅的感受性，並改變致病因子，包括運動性、生物膜形成，以及在壓力環境下的存活，例如抗生素下persistent cells形成。蠟蛾體內毒力試驗顯示hha缺失使毒力下降。同時我們發現hha 對致病因子的表達有dose effect的現象。Hha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。檢測纖毛相關基因顯示Hha和pmpA以及mrp表現呈正相關，而由細胞貼附試驗結果也發現hha突變株貼附尿上皮細胞能力較野生株下降。由抗壓相關表現型可觀察到hha突變株於酸性環境中與巨噬細胞試驗之生存率及persister cell的形成能力較野生株下降，同時觀察到hha突變株會使spoT、relA及rpoS等壓力相關基因表現減少。接著觀察到 hha突變株較易促使細胞激素分泌。其後探討了hha突變株的基因調控，tomB-hha operon具autoregulation， hha缺失引起調控外膜壓力的rpoE、rcsB及cueR 表現量下降。在缺失cueR及cpxR的情況下，hha 表現量則下降，表明cueR及cpxR參與 Hha 的調控路徑。最後我們發現銅會誘導hha 表現。由上述可得知，hha於P. mirabilis致病力及抗銅中扮演著重要的角色。 這是首次發現TA系統toxin參與尿道致病菌P. mirabilis致病因子表現的研究。

Proteus mirabilis, a Gram-negative bacterium, is an important urinary tract pathogen in human, often causing urinary tract infections (UTI). Copper is a member of host innate immune system. When a urinary tract infection occurs, the copper concentration of human urine will increase, together with the copper increase in macrophages, to protect from the invading pathogens. Because the accumulation of excessive copper causes damages to the bacterial cells, the bacteria evolve a series of anti-copper mechanisms to maintain the copper homeostasis. To investigate copper resistance mechanisms in P. mirabilis, we isolated copper-sensitive mutants by transposon mutagenesis. One of the mutant was found to be disrupted in the hha gene. Hha and TomB constitute a toxin-antitoxin system (TA) in Escherichia coli and Salmonella in which the antitoxin TomB is an enzyme which modifies the Hha toxin directly. Studies performed in E. coli and Salmonella also highlight the contribution of Hha in regulating bacterial virulence. Therefore, we investigated the role of Hha in virulence factor expression and found that hha mutant had a significant decrease in swimming, swarming and biofilm-forming ability. The hha mutant also exhibited a higher copper susceptibility than the wild-type strain with a dose-effect. It means that only within an optimal concentration range of Hha could restore the mutant phenotype to the wild type strain. The virulence test of Galleria mellonella showed that the loss of hha decreased the virulence compared with wild-type and complemented strain. The reporter assay revealed that Hha positively regulated flhDC promoter activity. Moreover, the expression of the infectivity-related fimbrial genes, mrpA and pmpA, in hha mutant was significantly decreased. The hha mutant had lower spoT, relA and rpoS (genes involved in coping with stress environment) expression by the reporter assay. Accordingly, we observed that hha mutant had a lower survival rate on exposure to low pH or in macrophages and a lower ability to form persistent cells than the wild-type. The hha mutant exhibited lower adhesion ability and induced higher cytokine production than the wild-type strain. The expression of rpoE, rcsB and cueR was reduced in hha mutant, which suggests hha may participate in the rpoE, rcsB and cueR regulated pathways. We identified that copper or urea (high osmolarity) acts as a signal for induction of Hha expression, indicating the triggering of Hha-transduction pathway. In conclusion, Hha plays an important role in copper susceptibility and pathogenicity of P. mirabilis. This is the first study of investigating the TA system in P. mirabilis.