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研究生(外文):Yi-Hwa Chou
論文名稱(外文):Type VI secretion system in uropathogenicProteus mirabilis N2
外文關鍵詞:P. mirabilisType VI secretion systemDienes linekilling
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Proteus mirabilis是造成泌尿道感染的病原菌之一,主要在長期使用尿導管的病人中造成伺機性感染。為了存活,細菌發展出許多調控機制,來適應環境中各種變化。第六型分泌系統(T6SS)是細菌間用來彼此競爭的武器之一,除了在同種及不同種菌之間的競爭外,T6SS亦會作用在真核細胞上。先前文獻指出,T6SS會受到與環境相關的regulators所調控,且T6SS所分泌的effector proteins (toxins)的種類及功能有很多,原核及真核細胞都可能為其作用標的。但有關T6SS在P. mirabilis當中的調控情形及作用尚待釐清,因此本研究利用P. mirabilis的regulators及sigma factors突變株來探討其對T6SS的影響。
從transcriptome dRNA-seq的結果發現,Crp、Hfq、RpoN、RpoE 及CpxR會影響P. mirabilis N2的T6SS相關基因的表現。透過swarming assay發現野生株會跟上述突變株形成boundary(Dienes line),辨認為不同菌株。利用生長優勢實驗發現野生株皆較具生長優勢。接著透過短時間殺菌試驗觀察到突變株都會被野生株所殺。進一步利用real-time RT-PCR及promoter reporter assay證明Crp及Hfq會正向調控T6SS結構基因。而T6SS會利用hcp-vgrG effector operons執行其功能,在這方面,Crp、Hfq、RpoN、RpoE 及CpxR都會正向調控各套hcp-vgrG effector operons。進一步探討Crp對T6SS的調控,透過crp互補株再次驗證Crp確實會正向調控T6SS結構基因及各套effector operons,也利用DNase I footprinting證明Crp會直接結合在T6SS結構基因及各套effector operons的啟動子區域,並透過殺菌試驗發現crp過度表現株具有較強的殺菌能力。在toxin的部分,我們發現Crp也會影響T6SS中三個可能的toxin gene的表現,其中2040及1472過度表現株使其對P. mirabilis及E. coli均展現了較強的殺菌能力。
綜合上述結果我們認為Crp、Hfq、RpoN、RpoE 及CpxR皆會調控P. mirabilis
N2 T6SS的表現,其中以Crp的影響最為顯著。此外,過度表現Crp及T6SS相關toxins會使其展現較強的殺菌能力。
Proteus mirabilis is a frequent pathogens causing urinary tract infection, mainly in patients with the long-term use of urinary catheters. Bacteria have developed diverse regulatory mechanisms for adaptation to the changing environments. Type VI secretion systm (T6SS) is one of the weapons for different bacteria to compete with each other and gain predominance in their niches. It is a protein secretion system which requires cell-cell contact, between bacteria or a bacterium and a eukaryotic cell. Studies have shown that T6SS is regulated by environment-related regulators, but the regulation of T6SS in P. mirabilis remains uncleared. Our transcriptome analysis showed T6SS of P. mirabilis N2 including T6SS main structure gene and four hcp-vgrG effector operons is likely regulated by Crp, Hfq, CpxR, RpoN and RpoE. We observed dienes lines formation between wild-type and crp, hfq, cpxR, rpoN and rpoE mutant strains. The wild-type has growth predomince over all mutant strains and all mutant strains are killed by the wild-type. It means all above regulators and sigma factors affect killing ability of P. mirabilis. We found the P. mirabilis genome contains one T6SS main structure (17 genes) operon and four T6SS hcp-vgrG effector operons by sequence specific reverse transcription PCR. Realtime PCR and promoter reporter assay demonstrated expression of T6SS main structure and/or four hcp-vgrG effector were up-regulated by Crp, Hfq, CpxR, RpoN and RpoE. We further found that Crp directly binds to the promoter regions of T6SS structure and four effector operons by DNase I footprinting analysis. In addition, crp-overexpression strain showed better killing ability against the wild-type. We used bioinformatic tools and found 3 putative toxin genes in T6SS of P. mirabilis N2. Toxin-overexpression strain also exhibited better killing ability than the wild-type and E. coli. Altogether, our study suggests that Crp, Hfq, CpxR, RpoN and RpoE positively regulated T6SS of P. mirabilis N2 and affected its killing ability.
誌謝 I
摘要 II
目錄 IV
表目錄 VI
圖目錄 VII
第一章 緒論 1
第一節 奇異變形桿菌(Proteus mirabilis)簡介 1
第二節 奇異變形桿菌的毒性因子 1
第三節 奇異變形桿菌的表面移行能力及其調控 3
第四節 第六型分泌系統(Type VI Secretion System, T6SS) 4
第五節 Crp、Hfq、RpoN、RpoE及 CpxR簡介 8
第六節 研究動機與目的 12
第二章 實驗材料與方法 13
第一節 實驗流程 13
第二節 實驗材料 14
第三節 質體構築 20
第四節 分析P. mirabilis野生株及突變株之間的競爭 30
第五節 基因表達 37
第三章 實驗結果 44
第一節 P. mirabilis N2的T6SS及各套hcp-vgrG effector operons 44
第二節 P.mirabilis N2野生株及突變株的競爭 51
第三節 P.mirabilis N2中T6SS及hcp-vgrG effector operons的調控方式 58
第四節 Crp對T6SS及hcp-vgrG effector operons的調控 67
第四章 結論與討論 77
第一節 結論 77
第二節 討論 78
第三節 未來展望 84
附錄 85
參考文獻 95
1.Rozalski A, Sidorczyk Z, Kotelko K. 1997. Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 61:65-89.
2.Foris L, Snowden J. 2017. Proteus Mirabilis Infections, StatPearls. StatPearls Publishing StatPearls Publishing LLC., Treasure Island (FL).
3.Schaffer JN, Pearson MM. 2015. Proteus mirabilis and Urinary Tract Infections. Microbiology spectrum 3:10.1128/microbiolspec.UTI-0017-2013.
4.Armbruster CE, Mobley HL. 2012. Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol 10:743-54.
5.Mobley HL, Belas R, Lockatell V, Chippendale G, Trifillis AL, Johnson DE, Warren JW. 1996. Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun 64:5332-40.
6.Mobley HL, Chippendale GR, Swihart KG, Welch RA. 1991. Cytotoxicity of the HpmA hemolysin and urease of Proteus mirabilis and Proteus vulgaris against cultured human renal proximal tubular epithelial cells. Infect Immun 59:2036-42.
7.Jacobsen SM, Shirtliff ME. 2011. Proteus mirabilis biofilms and catheter-associated urinary tract infections. Virulence 2:460-5.
8.Coker C, Poore CA, Li X, Mobley HL. 2000. Pathogenesis of Proteus mirabilis urinary tract infection. Microbes Infect 2:1497-505.
9.Drechsel H, Thieken A, Reissbrodt R, Jung G, Winkelmann G. 1993. Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases. J Bacteriol 175:2727-33.
10.Harshey RM. 2003. Bacterial motility on a surface: many ways to a common goal. Annu Rev Microbiol 57:249-73.
11.Rauprich O, Matsushita M, Weijer CJ, Siegert F, Esipov SE, Shapiro JA. 1996. Periodic phenomena in Proteus mirabilis swarm colony development. J Bacteriol 178:6525-38.
12.Givskov M, Ostling J, Eberl L, Lindum PW, Christensen AB, Christiansen G, Molin S, Kjelleberg S. 1998. Two separate regulatory systems participate in control of swarming motility of Serratia liquefaciens MG1. J Bacteriol 180:742-5.
13.Busby S, Ebright RH. 1999. Transcription activation by catabolite activator protein (CAP). J Mol Biol 293:199-213.
14.Weickert MJ, Adhya S. 1993. The galactose regulon of Escherichia coli. Mol Microbiol 10:245-51.
15.Bott M. 1997. Anaerobic citrate metabolism and its regulation in enterobacteria. Arch Microbiol 167:78-88.
16.Peterkofsky A, Gazdar C. 1974. Glucose inhibition of adenylate cyclase in intact cells of Escherichia coli B. Proc Natl Acad Sci U S A 71:2324-8.
17.Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH. 2004. Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol 14:10-20.
18.Savery N, Rhodius V, Busby S. 1996. Protein-protein interactions during transcription activation: the case of the Escherichia coli cyclic AMP receptor protein. Philos Trans R Soc Lond B Biol Sci 351:543-50.
19.Niu W, Kim Y, Tau G, Heyduk T, Ebright RH. 1996. Transcription Activation at Class II CAP-Dependent Promoters: Two Interactions between CAP and RNA Polymerase. Cell 87:1123-1134.
20.Beatty CM, Browning DF, Busby SJ, Wolfe AJ. 2003. Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism. J Bacteriol 185:5148-57.
21.Franze de Fernandez MT, Eoyang L, August JT. 1968. Factor fraction required for the synthesis of bacteriophage Qbeta-RNA. Nature 219:588-90.
22.Wang MC, Chien HF, Tsai YL, Liu MC, Liaw SJ. 2014. The RNA chaperone Hfq is involved in stress tolerance and virulence in uropathogenic Proteus mirabilis. PLoS One 9:e85626.
23.Aiba H. 2007. Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol 10:134-9.
24.Chao Y, Vogel J. 2010. The role of Hfq in bacterial pathogens. Curr Opin Microbiol 13:24-33.
25.Kazmierczak MJ, Wiedmann M, Boor KJ. 2005. Alternative Sigma Factors and Their Roles in Bacterial Virulence. Microbiology and Molecular Biology Reviews 69:527-543.
26.Zhang N, Buck M. 2015. A perspective on the enhancer dependent bacterial RNA polymerase. Biomolecules 5:1012-9.
27.Reitzer L. 2003. Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol 57:155-76.
28.Helmann JD. 2002. The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol 46:47-110.
29.Rowley G, Spector M, Kormanec J, Roberts M. 2006. Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens. Nat Rev Microbiol 4:383-94.
30.Hayden JD, Ades SE. 2008. The extracytoplasmic stress factor, sigmaE, is required to maintain cell envelope integrity in Escherichia coli. PLoS One 3:e1573.
31.Raivio TL, Silhavy TJ. 1997. Transduction of envelope stress in Escherichia coli by the Cpx two-component system. J Bacteriol 179:7724-33.
32.Pogliano J, Lynch AS, Belin D, Lin EC, Beckwith J. 1997. Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev 11:1169-82.
33.Snyder WB, Davis LJ, Danese PN, Cosma CL, Silhavy TJ. 1995. Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of periplasmic LacZ by activation of the Cpx signal transduction pathway. J Bacteriol 177:4216-23.
34.Danese PN, Snyder WB, Cosma CL, Davis LJ, Silhavy TJ. 1995. The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP. Genes Dev 9:387-98.
35.Alteri CJ, Himpsl SD, Pickens SR, Lindner JR, Zora JS, Miller JE, Arno PD, Straight SW, Mobley HL. 2013. Multicellular bacteria deploy the type VI secretion system to preemptively strike neighboring cells. PLoS Pathog 9:e1003608.
36.Russell AB, Singh P, Brittnacher M, Bui NK, Hood RD, Carl MA, Agnello DM, Schwarz S, Goodlett DR, Vollmer W, Mougous JD. 2012. A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11:538-49.
37.Alteri CJ, Mobley HL. 2016. The Versatile Type VI Secretion System. Microbiol Spectr 4.
38.Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, Heidelberg JF, Mekalanos JJ. 2006. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 103:1528-33.
39.Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordonez CL, Lory S, Walz T, Joachimiak A, Mekalanos JJ. 2006. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312:1526-30.
40.Bingle LE, Bailey CM, Pallen MJ. 2008. Type VI secretion: a beginner''s guide. Curr Opin Microbiol 11:3-8.
41.Pukatzki S, Ma AT, Revel AT, Sturtevant D, Mekalanos JJ. 2007. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A 104:15508-13.
42.Leiman PG, Basler M, Ramagopal UA, Bonanno JB, Sauder JM, Pukatzki S, Burley SK, Almo SC, Mekalanos JJ. 2009. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc Natl Acad Sci U S A 106:4154-9.
43.Ma J, Sun M, Dong W, Pan Z, Lu C, Yao H. 2016. PAAR-Rhs proteins harbor various C-terminal toxins to diversify the antibacterial pathways of type VI secretion systems. Environ Microbiol doi:10.1111/1462-2920.13621.
44.Sana TG, Flaugnatti N, Lugo KA, Lam LH, Jacobson A, Baylot V, Durand E, Journet L, Cascales E, Monack DM. 2016. Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut. Proc Natl Acad Sci U S A 113:E5044-51.
45.Silverman JM, Brunet YR, Cascales E, Mougous JD. 2012. Structure and regulation of the type VI secretion system. Annu Rev Microbiol 66:453-72.
46.Aschtgen MS, Gavioli M, Dessen A, Lloubes R, Cascales E. 2010. The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall. Mol Microbiol 75:886-99.
47.Felisberto-Rodrigues C, Durand E, Aschtgen MS, Blangy S, Ortiz-Lombardia M, Douzi B, Cambillau C, Cascales E. 2011. Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. PLoS Pathog 7:e1002386.
48.Zoued A, Brunet YR, Durand E, Aschtgen MS, Logger L, Douzi B, Journet L, Cambillau C, Cascales E. 2014. Architecture and assembly of the Type VI secretion system. Biochim Biophys Acta 1843:1664-73.
49.Aschtgen MS, Bernard CS, De Bentzmann S, Lloubes R, Cascales E. 2008. SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli. J Bacteriol 190:7523-31.
50.Ma LS, Lin JS, Lai EM. 2009. An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens. J Bacteriol 191:4316-29.
51.Ma LS, Narberhaus F, Lai EM. 2012. IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion. J Biol Chem 287:15610-21.
52.Cianfanelli FR, Monlezun L, Coulthurst SJ. 2016. Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon. Trends in Microbiology 24:51-62.
53.Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG. 2013. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500:350-3.
54.Cianfanelli FR, Alcoforado Diniz J, Guo M, De Cesare V, Trost M, Coulthurst SJ. 2016. VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System. PLoS Pathog 12:e1005735.
55.Busby JN, Panjikar S, Landsberg MJ, Hurst MR, Lott JS. 2013. The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device. Nature 501:547-50.
56.Koskiniemi S, Lamoureux JG, Nikolakakis KC, t''Kint de Roodenbeke C, Kaplan MD, Low DA, Hayes CS. 2013. Rhs proteins from diverse bacteria mediate intercellular competition. Proc Natl Acad Sci U S A 110:7032-7.
57.Willett JLE, Gucinski GC, Fatherree JP, Low DA, Hayes CS. 2015. Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways. Proceedings of the National Academy of Sciences of the United States of America 112:11341-11346.
58.Chou S, Bui NK, Russell AB, Lexa KW, Gardiner TE, LeRoux M, Vollmer W, Mougous JD. 2012. Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system. Cell Rep 1:656-64.
59.Whitney JC, Chou S, Russell AB, Biboy J, Gardiner TE, Ferrin MA, Brittnacher M, Vollmer W, Mougous JD. 2013. Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair. J Biol Chem 288:26616-24.
60.Russell AB, LeRoux M, Hathazi K, Agnello DM, Ishikawa T, Wiggins PA, Wai SN, Mougous JD. 2013. Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 496:508-12.
61.Unterweger D, Kostiuk B, Pukatzki S. 2017. Adaptor Proteins of Type VI Secretion System Effectors. Trends Microbiol 25:8-10.
62.Budding AE, Ingham CJ, Bitter W, Vandenbroucke-Grauls CM, Schneeberger PM. 2009. The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis. Journal of Bacteriology 191:3892-3900.
63.Bode NJ, Debnath I, Kuan L, Schulfer A, Ty M, Pearson MM. 2015. Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis. Infect Immun 83:2542-56.
64.Hood RD, Singh P, Hsu F, Guvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL, Li M, Schwarz S, Wang WY, Merz AJ, Goodlett DR, Mougous JD. 2010. A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7:25-37.
65.Brooks TM, Unterweger D, Bachmann V, Kostiuk B, Pukatzki S. 2013. Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB. J Biol Chem 288:7618-25.
66.Miyata ST, Kitaoka M, Brooks TM, McAuley SB, Pukatzki S. 2011. Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun 79:2941-9.
67.Aubert DF, Flannagan RS, Valvano MA. 2008. A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia. Infect Immun 76:1979-91.
68.Moscoso JA, Mikkelsen H, Heeb S, Williams P, Filloux A. 2011. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ Microbiol 13:3128-38.
69.Chakraborty S, Sivaraman J, Leung KY, Mok YK. 2011. Two-component PhoB-PhoR regulatory system and ferric uptake regulator sense phosphate and iron to control virulence genes in type III and VI secretion systems of Edwardsiella tarda. J Biol Chem 286:39417-30.
70.Salomon D, Gonzalez H, Updegraff BL, Orth K. 2013. Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2. PLoS One 8:e61086.
71.Pieper R, Huang ST, Robinson JM, Clark DJ, Alami H, Parmar PP, Perry RD, Fleischmann RD, Peterson SN. 2009. Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis. Microbiology 155:498-512.
72.Bernard CS, Brunet YR, Gavioli M, Lloubes R, Cascales E. 2011. Regulation of type VI secretion gene clusters by sigma54 and cognate enhancer binding proteins. J Bacteriol 193:2158-67.
73.Zheng J, Ho B, Mekalanos JJ. 2011. Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae. PLoS One 6:e23876.
74.Navarre WW, McClelland M, Libby SJ, Fang FC. 2007. Silencing of xenogeneic DNA by H-NS-facilitation of lateral gene transfer in bacteria by a defense system that recognizes foreign DNA. Genes Dev 21:1456-71.
75.Lucchini S, Rowley G, Goldberg MD, Hurd D, Harrison M, Hinton JC. 2006. H-NS mediates the silencing of laterally acquired genes in bacteria. PLoS Pathog 2:e81.
76.Castang S, McManus HR, Turner KH, Dove SL. 2008. H-NS family members function coordinately in an opportunistic pathogen. Proc Natl Acad Sci U S A 105:18947-52.
77.Wu CF, Lin JS, Shaw GC, Lai EM. 2012. Acid-induced type VI secretion system is regulated by ExoR-ChvG/ChvI signaling cascade in Agrobacterium tumefaciens. PLoS Pathog 8:e1002938.
78.Gode-Potratz CJ, McCarter LL. 2011. Quorum sensing and silencing in Vibrio parahaemolyticus. J Bacteriol 193:4224-37.
79.Shalom G, Shaw JG, Thomas MS. 2007. In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages. Microbiology 153:2689-99.
80.Schweizer HP, Hoang TT. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15-22.
81.Jiang SS, Lin TY, Wang WB, Liu MC, Hsueh PR, Liaw SJ. 2010. Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance, swarming, and virulence. Antimicrob Agents Chemother 54:2000-9.
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