鄧麗珍臺灣大學:醫學檢驗暨生物技術學研究所林詮盛Lin, Chuan-ShengChuan-ShengLin2010-05-112018-07-062010-05-112018-07-062008U0001-0407200815524800http://ntur.lib.ntu.edu.tw//handle/246246/182935Serratia marcescens是一隻很重要的伺機性感染的病原菌，有關其致病機制的研究仍舊有限。在我們之前的研究已發現到一套二元系統(two component system) RssA-RssB (RssAB)負向調控S. marcescens表面移行(swarming)行為，表面移行在許多病原菌中都已指出與致病因子的表現和病徵有相關性。因此，我們想探討RssAB如何調控S. marcescens的致病機轉。在本篇研究中，首先利用大鼠急性肺炎模式發現rssBA基因被剔除時會導致S. marcescens變成高度致病性的菌株，不管是致死率和病理特徵都明顯比野生株來的嚴重。進一步去探討之中的機制，RssAB訊息傳遞對於早期生物膜的形成是必須的。在野生株中，RssAB可以經由抑制flhDC的基因表現，進而壓抑S. marcescens重要的毒性因子-溶血素(hemolysin/ ShlA)的表現，利用in vitro的方式，rssBA基因剔除突變株(ΔrssBA)不管是對於綿羊紅血球的溶血能力、或者對於人類氣管表皮細胞(BEAS-2B)的毒殺與侵犯能力都明顯比野生株來的高。在未達致死菌量的大鼠肺炎模式中我們進一步發現ΔrssBA能夠導致全身性的感染，但野生株卻在肺部就有效被抑制住而無法侵犯深層組織，並在ΔrssBA中將溶血素基因shlBA剔除時就失去侵犯的能力，進而證明RssAB確實透過調控溶血素的表現而影響S. marcescens的致病能力。我們提出一個機制，RssAB二元系統在野生株中原本是扮演一個「毒性抑制」(antivirulence)的角色，調控表面移行與生物膜等不同的多細胞行為，以利適應不同的環境，進而控制S. marcescens 的致病能力與病理結果。未來的研究重點放在另一方面，我們想利用結合綠色螢光蛋白的RssAB，來探討在S. marcescens in vitro BEAS-2B模式與in vivo動物感染的各個階段與部位，RssAB會有怎樣的活化表現，並從此結果中去推測RssAB可能感受的訊息物質或環境會是什麼，從而了解S. marcescens的致病機轉。Serratia marcescens, as an important opportunistic pathogen, presents different multicellular behaviors like swarming and biofilm and possesses many virulence factors to adapt to diverse environments. However, the underlying mechanism of coordinating multicellularity, virulence expression, and pathogenesis of S. marcescens is unclear. Here, we show that two component system RssAB acts as an antivurlence modulator and inactivation of rssBA leads to hypervirulence phenotype of S. marcescens compared with wild type strain in acute pneumonia model of rat. Furthermore, RssAB inversely regulates swarming motility and early biofilm formation accompanied with contrary of expression of dominant virulence factor hemolysin ShlA. Associated with precocious swarming and defect in early biofilm formation, deletion of rssBA causes S. marcescens elevated hemolysin production concomitant with rising cytotoxicity and invasion against to human bronchial epithelial cell owing to derepression of flhDC in transcription level. Furthermore, in sublethal pneumonia model, we find that RssAB determine the capability of S. marcscens to cause systemic infection through modulating hemolysin. Without RssAB, this fine tuning in host-pathogen balance will lose and be toward to hypervirulent phenotypes during S. marcescens infection. We propose that S. marcescens utilizes RssAB to coordinate different multicellular behaviors and moderate virulence factor expression.Contents文摘要 ibstract iiontents iiiigure and Table contents vihapter 1 Introduction .1 S. marcescens is an important opportunistic pathogen 1.2 The regulation of dominant virulence factor hemolysin ShlBA is still unclear n S. marcescens 1.3 Two component system RssAB was involved in the modulation of virulence of. marcescens 2.4 Aim and strategy of this study 2hapter 2 Materials & Methods .1 Bacteria strains, plasmids, and primers 5.2 Bacteria medium and growth conditions 7.3 Enzymes, chemicals, and reagents 7.4 DNA technique 8.4.1 Isolation of plasmid DNA 8.4.2 Preparation of bacterial chromosomal DNA 8.4.3 Purification of DNA from agarose gels 9.4.4 Construction of recombinant plasmids 10.4.5 Transformation 10.4.6 Electroporation 11.4.7 Blue-white screening for recombinant plasmids 12.4.8 Colony-PCR screening for recombinant plasmids 13.4.9 Southern blot analyses 13.4.10 Plasmid transfer from E. coli S17-1 to S. marcescens CH-1 by conjugation 14.4.11 Construction of shlBA deletion mutants 15.4.12 Construction of pFlhDC 15.5 Biofilm attachment assay 16.6 RssAB signaling during biofilm formation 16.7 Hemolysis assay 17.8 Reverse transcription-PCR (RT-PCR) assay 17.9 Cell culture 18.10 Cellular cytotoxicity assay 18.11 Cellular invasion assay 19.12 Rat acute pneumonia model 21.13 Quantification of bacteria burden in lung and BAL fluid 21.14 Cell population analysis in BAL fluid. 22.15 Rat sublethal pneumonia model 22hapter 3 Results .1 Disruption of rssBA renders S. marcescens hypervirulent 24.2 RssAB signaling is required for early biofilm formation 27.3 RssAB represses hemolytic activity and shlBA gene expression through downregulating flhDC expression 28.4 Loss of rssBA promotes the cytotoxicity and invasion potency of S. marcescens into human bronchial epithelial cell BEAS-2B 31.5 Inactivation of rssBA renders S. marcescens the ability to cause systemic infection in the presence of hemolysin 34hapter 4 Discussions 38hapter 5 References 43ppendix Itandard buffers and solutions 48igure and Table contentsig. 1. Loss of rssBA promotes susceptibility of rats to S. marcescens in acute pneumonia model 27ig. 2. RssAB signaling controls the early biofilm formation in S. marcescens 29ig. 3. RssAB represses hemolytic activity and shlBA gene expression through downregulating flhDC expression 32ig. 4. Deletion of rssBA leads S. marcescens to elevated cytotoxic and invasive effect against to human bronchial epithelial cell BEAS-2B 35ig. 5. RssAB controls the outcome of pathogenesis in S. marcescens-induced sublethal pneumonia model of rat 37ig. 6. Proposed mechanism on how RssAB controls virulence and pathogenesis in S. marcescens 38able 1. Bacteria strains, plasmids, and primers used in this study 5application/pdf9668026 bytesapplication/pdfen-US表面移行生物膜多細胞行為溶血素致病能力Serratia marcescensswarmingbiofilmmulticellularityhemolysinvirulencepathogenesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182935/1/ntu-97-R95424004-1.pdf