廖淑貞Liaw, Shwu-Jen臺灣大學:醫學檢驗暨生物技術學研究所江信賢Jiang, Sin-SienSin-SienJiang2010-05-112018-07-062010-05-112018-07-062009U0001-1907200913304400http://ntur.lib.ntu.edu.tw//handle/246246/182972Proteus mirabilis屬革蘭氏陰性的腸內菌,是人類尿道感染的重要病原菌之一,特別在插導尿管的病人身上更容易發生感染。Antimicrobial peptides (APs),在動物及植物中屬先天性免疫的一部分,可在人類的中性球、巨噬細胞及表皮黏膜中發現。大部分的APs為帶正電,具雙極性的小分子胜肽,可非特異性的殺死外來致病菌。Polymyxin B (PB) 屬cationic antimicrobial peptides (CAMPs) 的一種,PB結構具有一帶多正電的polycationic peptide ring及一疏水性的尾端。CAMPs可以利用它所帶的正電與革蘭氏陰性菌LPS的lipid A或core部分上的負電做結合,進而造成膜的穿孔或瓦解。P. mirabilis天生就對PB具高抗性,其中之一的機制可能是透過lipid A上的4-amino-4-deoxy-L-arabinose (L-Ara4N) 修飾,此修飾亦在Escherichia.coli及Salmonella enteric serovar Typhimurium中被發現。在Salmonella中lipid A上的L-Ara4N修飾是透過PhoP-PhoQ及PmrA-PmrB兩套雙組成系統所調控;此修飾會降低LPS所帶的負電荷,造成減少PB的結合。實驗室在先前的研究中發現P. mirabilis中有PhoP-PhoQ的相似基因,並命名為RppA-RppB。而rppA的突變株對PB具高敏感性,此外也發現在低濃度的PB下rppA mRNA的表現量會增加。為了進一步探討P. mirabilis抗PB的機制,以mini-Tn5 轉位子突變法尋找其他與PB感受性相關的基因並進一步探討其可能的調控機制及觀察毒力因子的表現是否發生改變。 實驗發現三株對PB高敏感性的突變株,其MIC數值與野生株相差一萬倍以上,進一步發現轉位子所插入的基因分別為galU、pmrI相似物及PMI 1781。分析他們的LPS profile,發現galU和PM3突變株的LPS量很低且LPS的ladder也消失;而pmrI突變株的LPS profile與濃度則與野生株相似,但卻發現pmrI突變株的LPS與PB結合的能力較野生株佳。以原子力顯微鏡觀察也發現galU突變株的表面結構較野生株粗糙。而在PB的存在下,野生株的LPS會進行修飾造成LPS ladder產生位移,但在pmrI及rppA突變株中則無此現象。此外在PB下無法誘導rppA突變株但會誘導野生株galU及pmrI mRNA表現量近四倍的增加。由上述結果顯示,galU及pmrI基因可能受到RppA的調控,而pmrI亦與LPS的修飾有關。pmrI屬於pmrHFIJKLM operon中的一個基因,而由reporter assay及in vitro EMSA實驗進一步發現,RppA直接結合到pmrHFIJKLM 的promoter上。由此推測於PB存在下pmrI mRNA表現量的增加可能是透過RppA直接調控此operon所致。在表現型的分析方面,galU和PM3突變株 (LPS產生缺失的突變株),皆失去表面移行的能力,此外在galU突變株中與鞭毛合成的相關基因flhDC、 fliA及flaA的mRNA表現都下降。此外galU和PM3突變株的溶血酶活性、尿素酶活性及生物膜的合成都較野生株來的低。 綜合這些實驗結果可以發現,在P. mirabilis中,galU及PMI 1781基因會影響LPS的合成,而LPS的完整性不只影響PB感受性,還會影響表面移行能力及毒力因子的表現。此外LPS的修飾是透過pmrI基因所調控,而此修飾亦幫助P. mirabilis抑制PB的結合。最後,由實驗結果可證明P. mirabilis的RppA-RppB雙組成系統,在PB的存在下會去調控下游基因例如: galU及pmrI基因的表現。galU會影響LPS的合成而LPS的修飾則透過pmrI基因所調控,而此修飾則抑制了PB和P. mirabilis LPS的結合,進而達到抵抗PB的目的。Proteus mirabilis is a gram-negative bacterium and a member of the family Enterobacteriaceae. It is an important pathogen of the urinary tract, especially in patients with indwelling urinary catheters. Antimicrobial peptides (APs) are important components of the innate defenses of animals and plants, which can be found in neutrophils and macrophages and are produced by epithelial cells at mucosal surfaces. Most APs are cationic, amphipathic molecules of small molecule weight peptides and have an activity against infection of pathogens. Polymyxin B (PB), a kind of cationic antimicrobial peptides (CAMPs), is composed of a polycationic peptide ring and a hydrophobic tail. In gram-negative bacteria, CAMPs which have positive charge, can bind to negative charged portion of LPS such as lipid A and core and then alter the membrane integrity by solubilization or pore formation. P. mirabilis is naturally resistant to PB, and one of the possible mechanisms is through the modification of lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N) which is also found in Escherichia.coli and Salmonella enteric serovar Typhimurium. In Salmonella Typhimurium, L-Ara4N modification of lipid A is regulated by two-component systems, PhoP-PhoQ and PmrA-PmrB. The modification reduces the negative charge of LPS and consequently decreases the binding of PB. In our previous study, we found a PhoP-PhoQ homologue and designated RppA-RppB. The rppA knockout strain is highly sensitive to PB and the expression of rppA is up-regulated by a low concentration of PB. To better understand the underlying mechanisms of P. mirabilis resistance to PB, mini-Tn5 transposon mutagenesis is used to identify more genes involved in PB susceptibility and to characterize the function of these genes.hree unique PB-sensitive P. mirabilis mutants were identified and these mutants were over 10000-fold more sensitive than the wild-type. DNA sequence analysis of the transposon insertion gene reveals similarities to a galU, pmrI and PMI 1781 loci of E. coli and Salmonella. Though the galU and PM3 mutants have lower concentration of LPS and altered LPS profile than the wild-type, the pmrI mutant has normal LPS concentration and profile. Further, we found that the LPS of the pmrI mutant can bind more PB than the wild-type. The atomic force microscopy also showed that the surface structure of galU mutant is more roughness than wild-type. Moreover, in the presence of PB, LPS ladder occurs shifting in wild-type LPS profile but not in pmrI and rppA mutants, and the expression of galU and pmrI mRNA has four-fold increase. These data imply that galU and pmrI expression maybe regulated by response regulator RppA and pmrI is involved in LPS modification. Furthermore, the pmrI belongs to a pmrHFIJKLM operon, and we find that the promoter of this operon is enhanced by low concentration of PB, and we demonstrate that RppA protein can directly bind to the promoter to enhance this operon expression by EMSA experiment. When the mutants are streaked on 1.5% LB agar plate, the swarming motility of galU and PM3 mutants (LPS-defective mutants) are completely inhibited due to the defect in swarmer cell differentiation and decreased mRNA expression of flagellin biosynthesis genes (flhDC, fliA and flaA) in galU mutant. The other phenotypes such as haemolysin, urease activity and biofilm production are also decreased in galU and PM3 mutants. These data support the roles of galU and PMI 1781 in LPS synthesis, and LPS integrity plays an essential role in PB susceptibility, swarming and virulence factor expression in P. mirabilis. LPS modification, which is mediated by pmrI, also demonstrated to be the mechanism in the resistance of PB. Finally, our data also highlight the important role of RppA-RppB two-component system in the presence of PB to regulate downstream regulon, such as galU and pmrI and confer the ability against PB.誌謝 i目錄 ii目錄 (List of Tables) v目錄 (List of Figures) vi法目錄 (List of Procedures) viii文摘要 x文摘要 xii一章 緒論 1一節 奇異變形桿菌 (Proteus mirabilis) 介紹 1二節 多黏菌素B (polymyxin B, PB) 簡介及抗藥機轉介紹 5三節 研究動機與目的 9二章 實驗材料及流程 10一節 實驗材料 10二節 實驗流程與方法 11、 尋找對polymyxin B高敏感性的突變株 11、 分析突變株對polymyxin B的感受性:MIC test 11、 突變株的分析:觀察是否為單一轉位子插入及生長情形 11、 轉位子插入基因的選殖及定序 11、 突變株的脂多醣 (lipopolysaccharide, LPS) 分析 12、 分析轉位子所插入的基因是否受雙組成系統 (two-component system) 調控 12、 突變株的毒力因子表現分析 14、 突變株的補回 (complementation) 實驗 16三章 實驗結果 18一節 尋找對polymyxin B高敏感性的突變株並探討可能的抗藥機轉 18、 PM1、PM2、PM3突變株皆對polymyxin B具高感受性 18、 PM1、PM2、PM3突變株皆為單一轉位子插入,且不影響生長速率 18、 突變株中轉位子所插入的基因分別為galU、pmrI、PMI 1781 18、 pmrI、galU及PM3突變株的脂多醣 (lipopolysaccharide, LPS) 分析 20、 pmrI及galU基因的表現受RppA-RppB雙組成系統調控 21二節 探討polymyxin B感受性與毒力因子表現的關係 25、 表面移行 (swarming) 能力分析 25、 鞭毛蛋白 (flagellin) 分析 25、 細胞型態分析 26、 溶血酶 (haemolysin) 活性分析 26、 尿素酶 (urease) 活性分析 pmrI、galU及PM3突變株的尿素酶活性皆較野生株低。 27、 生物膜 (biofilm) 形成分析 27、 菌體表面特性分析 27、 細菌外膜蛋白 (outer membrane protein) 分析 28、 Real-time PCR分析與鞭毛蛋白合成相關的基因表現 28三節 突變株的補回 (complementation) 實驗 29、 galU突變株的complementation 29、 pmrI突變株的complementation 29四章 結果討論 30一節 galU、pmrI及PMI 1781基因與polymyxin B抗性的關係 30二節 galU、pmrI及PMI 1781基因與毒力因子表現的關係 32三節 galU及pmrI突變株的補回 33四節 總結與未來方向 34五章 表 35六章 圖 40七章 Procedures 58八章 參考文獻 92九章 附錄 98application/pdf15827063 bytesapplication/pdfen-US奇異變形桿菌多黏菌素B表面移行Proteus mirabilispolymyxinBswarming奇異變形桿菌中PmrI及GalU調控多黏菌素B的感受性、表面移行能力、毒力因子表現及其受RppA調控之研究Roles of PmrI and GalU in polymyxin B susceptibility, swarming, virulence factor expression and regulation of pmrI and galU by RppA in Proteus mirabilishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182972/1/ntu-98-R96424014-1.pdf