賴信志臺灣大學:醫學檢驗暨生物技術學研究所謝尚諶Hsieh, Shang-ChenShang-ChenHsieh2010-05-112018-07-062010-05-112018-07-062008U0001-1403200811054000http://ntur.lib.ntu.edu.tw//handle/246246/182932Swarming為一種細菌群體在固體表面移行之特殊型態，已知靈菌 (Serratia marcescens) 野生株CH-1在30 ºC時能在固體表面上進行swarming，但在37 ºC時則被抑制。為了解其中的機制，我們利用Tn5跳躍子篩選出能在37 ºC環境下進行swarming的突變株。其中之一突變株破壞在sspA基因的位置，經由序列比對後發現SspA為一全新的脂蛋白，而SspA的破壞可能與此一突變株在37 ºC swarming的行為有關。雖然尚未有直接而全盤的證據指出SspA在調控swarming的過程中所扮演的角色，但根據此一突變株的各種表現型，包括biosurfactant 產生的增加、鞭毛表現量的增加以及細胞附著能力的下降，可能都與SspA的破壞有關，進而導致sspA突變株在37 ºC下的swarming行為。除此之外，由於在突變株溶血素活性的上升，指出了swarming跟S. marcescens致病性的可能關聯。因此，我們另外對於S. marcescens 的致病機轉及其之所以為重要的伺機性病原菌的原因做進一步的探討。在我第二部份的研究發現，在大鼠模式中， S. marcescens的pyruvate代謝產物2,3-butanediol不但明顯抑制由細菌內毒素(脂多醣體)所誘發的急性肺損傷與相關的發炎反應，其效果幾乎與已知為一抗發炎多酚化合物葡萄籽萃取物resveratrol相當，進一步研究則發現2,3-butanediol能夠透過調節NF-κB的訊息傳遞路徑來抑制由脂多醣體所引起的急性發炎反應。因此，2,3-butanediol在細菌感染的過程中，對於宿主的innate免疫反應扮演著負向調控的角色，此結果也暗示了細菌利用其代謝產物逃避宿主免疫系統的可能性。Swarming in Serratia marcescens is a specialized form of bacterial surface migration. S. marcescens swarms at 30 ºC but not at 37 ºC on 0.8 % LB agar plate. To unravel the underlying mechanisms of the temperature-dependent swarming behavior, transposon mutagenesis was performed to screen for mutants that swarmed at 37 ºC. SspA, a novel lipoprotein, was identified to involve the negative regulation of S. marcescens swarming at 37 ºC. Increased production of biosurfactant, over-synthesis of flagellum and the reduced biofilm formation in sspA mutant S. marcescens SC101 all might contribute to the precocious-swarming behavior of S. marcescens SC101 at 37 ºC. Furthermore, the increased hemolytic activity of precocious-swarming mutant suggested that the regulation of swarming is closely related to the pathogenesis of S. marcescens. Besides, we also asked why S. marcescens is an important nosocomial pathogen. Herein, we showed that gastric intubation of 2,3-butanediol, a pyruvate metabolite produced by S. marcescens, in rats significantly ameliorates acute lung injury and the inflammatory responses induced by S. marcescens derived endotoxin lipopolysaccharide (LPS), with an efficacy comparable to that of the polyphenol compound resveratrol. Such effect was further demonstrated to occur via modulation of the NF-κB signaling pathway. Our results indicated that bacterial metabolite, 2,3-butanediol has a negative regulatory effect on host innate immunity response, suggesting bacteria may use some metabolites for host immune evasion.Contents 文摘要 ibstract iihapter 1 General Introduction 1hapter 2 Materials and Methods 25.1 Bacterial strains, plasmids, animals and primers 25.1.1 Bacterial strains 25.1.2 Plasmids 26.1.3 Animals and other chemicals 26.1.4 Primers 27.2 Materials and reagents 28.2.1 Kits 28.2.2 Enzymes and chemicals 28.2.3 Antibiotics 28.2.4 Media 29.2.5 DNA electrophoresis reagents 29.2.6 Southern blot hybridization reagents 30.2.7 Protein electrophoresis and Western blot analysis reagents 31.2.8 Plasmid purification reagents 32.2.9 Chromosomal DNA isolation reagents 32.2.10 General chemicals 33.2.11 Miscellaneous 34.2.12 Equipment and supplies 34.3 Experimental procedures 35.3.1 Isolation of plasmid DNA 35.3.2 Preparation of bacterial chromosomal DNA 36.3.3 Extraction of DNA from agarose gel 37.3.4 Restriction enzyme digestion 37.3.5 Ligation reaction 37.3.6 Calcium chloride transformation 37.3.6.1 Preparation of transformation-competent cells 37.3.6.2 Transformation of DNA 38.3.7 Electroporation 38.3.7.1 Preparation of electroporation -competent cells 38.3.7.2 Electroporation of DNA 39.3.8 Blue-white screening for recombinant plasmids 39.3.9 Components of PCR 40.3.9.1 Colony-PCR screening for recombinant plasmids 40.3.10 DNA sequence analysis and databank comparison 40.3.11 Plasmid transfer from E. coli S17-1 to S. marcescens by conjugation 41.3.12 Southern blot hybridization 41.3.12.1 Preparation of DNA probe 41.3.12.2 Transfer of digested DNA to membrane 42.3.12.3 Pre-hybridization and hybridization 42.3.12.4 DIG-detection assay 42.3.13 Strategies used to knock out sspA 43.3.14 Construction of recombinant plasmid for gene over-expression and complementation 44.3.15 Surface migration assay 44.3.15.1 Swarming assay of S. marcescens CH-1 44.3.15.2 Swimming assay of S. marcescens CH-1 44.3.16 Globomycin assay 44.3.17 Expression of recombinant proteins 45.3.17.1 SDS-PAGE 45.3.17.2 Western blot analysis 45.3.18 Drop-collapsing assay 46.3.19 Thin-layer chromatography (TLC) assay 46.3.20 Cell attachment assay 46.3.21 Assay of hemolytic activity 47.3.22 Induction of airway inflammation and drug pretreatment 47.3.23 Measurement of wet-to-dry weight ratio 47.3.24 BALF (bronchoaveolar lavage fluid) collection 48.3.25 Semi-quantification of BALF cellularity 48.3.26 Histological examination 48.3.27 Lung cytokine measurement 49.3.27.1 ELISA for lung cytokine protein 49.3.27.2 Reverse-transcription PCR for lung cytokine gene expression 49.3.28 NF-κB translocation assay 50.3.28.1 Immunocytochemistry stain 50.3.28.2 Western blot analysis for NF-κB subunit p65 51.3.28.3 NF-κB DNA binding assay 51.3.29 Data analysis 51hapter 3 SspA, a novel lipoprotein involved in regulation of swarming behavior in S. marcescens 52.1 Introduction 52.2 Results 54.2.1 Define a proper assay condition for isolation of precocious-swarming mutants 54.2.2 Characterization of the precocious-swarming phenotype of mini-Tn5 transposon mutant S. marcescens SC100 54.2.3 Characterization of S. marcescens SC100 precocious-swarming mutant locus 55.2.4 Construction of sspA insertion-deletion mutant S. marcescens SC101 derived from S. marcescens CH-1 57.2.5 Characterization of SC101 phenotypes 58.2.6 SspA is a novel outer membrane lipoprotein 62.3 Discussion 63hapter 4 Bacteria-host interaction: 2,3-butanediol, a bacterial carbohydrate metabolite, involved in amelioration of LPS-induced acute lung injury 79.1 Introduction 79.2 Results 81.2.1 2,3-butanediol attenuates LPS-induced lung edema 81.2.2 2,3-butanediol inhibits recruitment of neutrophils during acute inflammation phase 81.2.3 2,3-butanediol inhibits pro-inflammatory cytokine production in BALF 83.2.4 2,3-butanediol inhibits pro-inflammatory cytokine gene expression in lung tissue 83.2.5 2,3-butanediol inhibits NF-κB p65 nuclear translocation 84.2.6 2,3-butanediol inhibits LPS-dependent IκBα phosphorylation 86.3 Discussion 87hapter 5 Concluding discussion 100eference 104ppendix 122igure Contentsig. 3.1 Swarming of CH-1, SC100 and SC101 at 30 ºC and 37 ºC 69ig. 3.2 The transmembrane region of SspA 69ig. 3.3 Construction of sspA insertion-deletion mutant S. marcescens SC101 70ig. 3.4 Genetic maps of sspA knock-out mutant and primer designs for PCR screening 71ig. 3.5 Southern blot analysis for confirmation of SC101 genotype 72ig. 3.6 The growth curve of CH-1, SC100 and SC101 73ig. 3.7 The swarming velocity of CH-1, SC100 and SC101 73ig. 3.8 Complementation of sspA mutant at 37 ºC 74ig. 3.9 The swimming motility and flagella expression of CH-1 and SC101 at 37 ºC 75ig. 3.10 The biosurfactant produced by CH-1 and SC101 at 37 ºC 76ig. 3.11 Quantification of biofilm formation and hemolytic activity at 37 ºC 77ig. 3.12 Globomycin assay 78ig. 4.1 Neutrophil recruitment in rat lung tissue after LPS exposure is inhibited by 2,3-butanediol 94ig. 4.2 Production of pro-inflammatory cytokines is decreased by 2,3-butanediol 95ig. 4.3 Pro-inflammatory cytokine gene expression in lung tissue is reduced by 2,3-butanediol 96ig. 4.4 Immunohistochemical localization of NF-κB p65 in lung tissue 97ig. 4.5 NF-κB p65 nuclear translocation and activation are inhibited by 2,3-butanediol 98ig. 4.6 LPS-dependent IκBα phosphorylation is inhibited by 2,3-butanediol 99able contentsable 2.1 The bacteria strains used in this thesis 25able 2.2 The plasmids used in this thesis 26able 2.3 The primers used in this thesis 27able 4.1 Wet-to dry ratio of lung tissue 6 h after intratracheal LPS instillation 93able 4.2 BALF cellularity in rats 6 h after intratracheal LPS challenge 93application/pdf3869367 bytesapplication/pdfen-US靈菌脂蛋白表面移行脂多醣發炎S. marcescenslipoproteinswarming2,3-butanediolLPSinflammationhttp://ntur.lib.ntu.edu.tw/bitstream/246246/182932/1/ntu-97-F91424001-1.pdf