嗜水產氣單胞菌為一革蘭氏陰性菌，此菌不但是許多水生動物如魚類及變溫動物如爬蟲類、兩棲類等動物的病原菌以外，其亦可以感染陸上動物，包括造成人類患病。若受到嗜水產氣單胞菌的感染可能會造成腸胃炎或一些組織性的感染，嚴重者甚至引發敗血症。致病菌藉由複雜的基因調控來調節其致病機轉，而雙因子訊息傳遞系統在許多的致病菌中，似乎都扮演著在致病力上重要的角色，我們的目的是要了解其中一種雙因子訊息傳遞系統，此系統由兩個蛋白質所構成，CpxA 與CpxR，CpxA是一個具有組氨酸激酶活性的蛋白，而CpxR為一反應調控蛋白，當菌體受到外在的刺激時，便會啟動Cpx活化系統，進而將訊息傳遞給下游的基因，以因應該刺激所造成的變化。從過去的文獻發現，在大腸桿菌中，活化後的Cpx系統可以調控dsbA基因的轉錄效率與調控DsbA而提升五到十倍的表現量。DsbA可以催化受質蛋白形成雙硫鍵，其也可影響膜間隙蛋白和外泌性蛋白的活性。嗜水產氣單胞菌可分泌許多菌體外酵素，而這些菌體外酵素的活性可能藉由DsbA所調控，另外這些菌體外酵素也被認為可能是嗜水產氣單胞菌的致病因子，因此在本研究中，我們利用同源重組的原理，創造了CpxA與CpxR的基因突變株，並且觀察突變株與野生株的差異。從我們的結果得知，在DTT試驗、蛋白酶試驗和脂肪酶試驗中，突變株與野生株無顯著性的差異，然而在polymyxin B試驗中，在濃度20 µg/ml的培養之下，可在15及30分鐘培養的時間點，看到突變株與野生株有顯著性的差異，由此結果我們認為Cpx系統可能與菌體對抗polymyxin B的機制上有關聯性，另外在人類血清抗性試驗中，在濃度75%的培養之下，可在30分鐘培養的時間點，看到突變株與野生株有顯著性的差異。從人類血清抗性試驗所觀察到的結果似乎與polymyxin B試驗相仿，因此我們認為Cpx系統可能對於初始的外來壓力能感應並做出反應。然而，老鼠半致死率的試驗，突變株與野生株無顯著的差異。最後，我們認為Cpx雙因子訊息傳遞系統在嗜水產氣單胞菌的致病力上可能扮演著較次要性的角色，而在嗜水產氣單胞菌中，cpx被破壞之後，也不會影響DsbA的表現量。

Aeromonas hydrophila is a gram-negative bacterium which can cause disease in wide variety of waterborne animals, including reptiles, amphibians, and fish. It is also an emerging human pathogens that cause a wide array of diseases, such as gastroenteritis, wound infections, and septicemia. Since two-component systems are assumed to be one of the prime bacterial systems and likely play a major role in bacterial virulence, one of our purpose is to investigate whether one of two-component systems, CpxA and CpxR, in A. hydrophila is involved in the pathogenesis of this bacterium or not. Previous studies reported that CpxA is a histidine kinase sensor protein and CpxR is a response regulatory protein and CpxA/R system controls transcription of the dsbA locus and activation of the Cpx pathway in 5- to 10-fold the synthesis of DsbA(disulfide bond formation protein A) in Escherichia coli. Since DsbA is a oxidative catalyst in the periplasm and it affects disulfide bond formation of secretory and periplasmic proteins and since A. hydrophila produces several extracellular proteins whose disulfate-bond formation may be catalyzed by DsbA and these extracellular proteins may act as the virulence factors. In this study, we constructed cpxA isogenic mutant and cpxR isogenic mutant by homologous recombination, and determined the properties of these two isogenic mutant. In our data, no significantly difference in DTT(dithiothreitol) assay, extracellular proteaolytic and lipolytic activities between two isogenic mutant and wild type strain was observed. However, both the two isogenic mutant exhibited significantly difference lower survival than the wild-type strain when incubated for 15 and 30 min in LB broth containing 20 µg/ml polymyxin B. This findings suggest that the CpxA/R system may be required for resistant to polymyxin B. After incubating with 75% human serum for 30 min, the survival rates of both isogenic mutant strains were shown to be significantly lower than wild type strain. The appearance in human serum-resistant assay match with polymyxin B assay was observed.We suggest that CpxA/R system could be make the response in undergoing stress at early stage. However, the value LD50 of mice infected by the two isogenic mutant via intraperitioneal was not significantly difference with wild type. Finally, we suggest that cpx two-component system was played a less important role in pathogenesis of Aeromonas hydrophila. However, expression of DsbA did not influenced by interruption of cpx in Aeromonas hydrophila.

Abrami L, Fivaz M, Glauser PE, Sugimoto N, Zurzolo C, van der Goot FG (2003) Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin. Infect Immun 71: 739-746
Andersen CL, Matthey-Dupraz A, Missiakas D, Raina S (1997) A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins. Mol Microbiol 26: 121-132
Austin B (1996) The genus aeromonas. J. Wiley Inc., New York
Bader MW, Hiniker A, Regeimbal J, Goldstone D, Haebel PW, Riemer J, Metcalf P, Bardwell JC (2001) Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA. EMBO J 20: 1555-1562
Bader MW, Xie T, Yu CA, Bardwell JC (2000) Disulfide bonds are generated by quinone reduction. J Biol Chem 275: 26082-26088
Bardwell JC, Lee JO, Jander G, Martin N, Belin D, Beckwith J (1993) A pathway for disulfide bond formation in vivo. Proc Natl Acad Sci USA 90: 1038-1042
Bardwell JC, McGovern K, Beckwith J (1991) Identification of a protein required for disulfide bond formation in vivo. Cell 67: 581-589
Bessette PH, Cotto JJ, Gilbert HF, Georgiou G (1999) In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG. J Biol Chem 274: 7784-7792
Brumlik MJ, van der Goot FG, Wong KR, Buckley JT (1997) The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity. J Bacteriol 179: 3116-3121
Canals R, Jimenez N, Vilches S, Regue M, Merino S, Tomas JM (2006) The UDP N-acetylgalactosamine 4-epimerase gene is essential for mesophilic Aeromonas hydrophila serotype O34 virulence. Infect Immun 74: 537-548
Carmel-Harel O, Storz G (2000) Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol 54: 439-461
Cascon A, Yugueros J, Temprano A, Sanchez M, Hernanz C, Luengo JM, Naharro G (2000) A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila. Infect Immun 68: 3233-3241
Chang C, Stewart RC (1998) The two-component system. Regulation of diverse signaling pathways in prokaryotes and eukaryotes. Plant Physiol 117: 723-731
Chang MC, Chang SY, Chen SL, Chuang SM (1992) Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease (DNase) from Aeromonas hydrophila. Gene 122: 175-180
Cheng NC, Horng SY, Chang SC, Tang YB (2004) Nosocomial infection of Aeromonas hydrophila presenting as necrotizing fasciitis. J Formos Med Assoc 103: 53-57
Chuang YC, Chiou SF, Su JH, Wu ML, Chang MC (1997) Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2. Microbiology 143: 803-812
Couprie J, Vinci F, Dugave C, Qu inverted question markem inverted question markeneur E, Moutiez M (2000) Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex. Biochemistry 39: 6732-6742
Dailey FE, Berg HC (1993) Mutants in disulfide bond formation that disrupt flagellar assembly in Escherichia coli. Proc Natl Acad Sci USA 90: 1043-1047
Danese PN, Silhavy TJ (1997) The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli. Genes Dev 11: 1183-1193
Dodson KW, Pinkner JS, Rose T, Magnusson G, Hultgren SJ, Waksman G (2001) Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor. Cell 105: 733-743
Duthie R, Ling TW, Cheng AF, French GL (1995) Aeromonas septicaemia in Hong Kong species distribution and associated disease. J Infect 30: 241-244
Epple HJ, Mankertz J, Ignatius R, Liesenfeld O, Fromm M, Zeitz M, Chakraborty T, Schulzke JD (2004) Aeromonas hydrophila beta-hemolysin induces active chloride secretion in colon epithelial cells (HT-29/B6). Infect Immun 72: 4848-4858
Erova TE, Pillai L, Fadl AA, Sha J, Wang S, Galindo CL, Chopra AK (2006) DNA adenine methyltransferase influences the virulence of Aeromonas hydrophila. Infect Immun 74: 410-424
Esteve C, Birbeck TH (2004) Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (elastase). J Appl Microbiol 96: 994-1001
Fang HM, Ge R, Sin YM (2004) Cloning, characterisation and expression of Aeromonas hydrophila major adhesin. Fish Shellfish Immunol 16: 645-658
Favre D, Ngai PK, Timmis KN (1993) Relatedness of a periplasmic, broad-specificity RNase from Aeromonas hydrophila to RNase I of Escherichia coli and to a family of eukaryotic RNases. J Bacteriol 175: 3710-3722
Funada H, Matsuda T (1997) Aeromonas bacteremia in patients with hematologic diseases. Intern Med 36: 171-174
Gobius KS, Pemberton JM (1988) Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila. J Bacteriol 170: 1325-1332
Gold WL, Salit IE (1993) Aeromonas hydrophila infections of skin and soft tissue: report of 11 cases and review. Clin Infect Dis 16: 69-74
Heras B, Edeling MA, Schirra HJ, Raina S, Martin JL (2004) Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide. Proc Natl Acad Sci USA 101: 8876-8881
Hiniker A, Bardwell JC (2004) In vivo substrate specificity of periplasmic disulfide oxidoreductases. J Biol Chem 279: 12967-12973
Humphreys S, Rowley G, Stevenson A, Anjum MF, Woodward MJ, Gilbert S, Kormanec J, Roberts M (2004) Role of the two-component regulator CpxAR in the virulence of Salmonella enterica serotype Typhimurium. Infect Immun 72: 4654-4661
Isaac DD, Pinkner JS, Hultgren SJ, Silhavy TJ (2005) The extracytoplasmic adaptor protein CpxP is degraded with substrate by DegP. Proc Natl Acad Sci USA 102: 17775-17779
Jander G, Martin NL, Beckwith J (1994) Two cysteines in each periplasmic domain of the membrane protein DsbB are required for its function in protein disulfide bond formation. EMBO J 13: 5121-5127
Jones CH, Danese PN, Pinkner JS, Silhavy TJ, Hultgren SJ (1997) The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems. EMBO J 16: 6394-6406
Kadokura H, Katzen F, Beckwith J (2003) Protein disulfide bond formation in prokaryotes. Annu Rev Biochem 72: 111-135
Ko WC, Chuang YC (1995) Aeromonas bacteremia: review of 59 episodes. Clin Infect Dis 20: 1298-1304
Ko WC, Lee HC, Chuang YC, Liu CC, Wu JJ (2000) Clinical features and therapeutic implications of 104 episodes of monomicrobial Aeromonas bacteraemia. J Infect 40: 267-273
Ko WC, Lee HC, Chuang YC, Ten SH, Su CY, Wu JJ (2001) In vitro and in vivo combinations of cefotaxime and minocycline against Aeromonas hydrophila. Antimicrob Agents Chemother 45: 1281-1283
Ko WC, Wu HM, Chang TC, Yan JJ, Wu JJ (1998) Inducible beta-lactam resistance in Aeromonas hydrophila: therapeutic challenge for antimicrobial therapy. J Clin Microbiol 36: 3188-3192
Lan X, Ozawa N, Nishiwaki N, Kodaira R, Okazaki M, Shimosaka M (2004) Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9. Biosci Biotechnol Biochem 68: 1082-1090
Lan X, Zhang X, Hu J, Shimosaka M (2006) Cloning, expression, and characterization of a chitinase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9. Biosci Biotechnol Biochem 70: 2437-2442
Lin CS, Cheng SH (1998) Aeromonas hydrophila sepsis presenting as meningitis and necrotizing fasciitis in a man with alcoholic liver cirrhosis. J Formos Med Assoc 97: 498-502
Mallick P, Boutz DR, Eisenberg D, Yeates TO (2002) Genomic evidence that the intracellular proteins of archaeal microbes contain disulfide bonds. Proc Natl Acad Sci USA 99: 9679-9684
Martin JL, Bardwell JC, Kuriyan J (1993) Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature 365: 464-468
McCarthy AA, Haebel PW, Torronen A, Rybin V, Baker EN, Metcalf P (2000) Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli. Nat Struct Biol 7: 196-199
Merino S, Rubires X, Aguilar A, Tomas JM (1997) The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains. FEMS Microbiol Lett 151: 213-217
Messens J, Collet JF (2006) Pathways of disulfide bond formation in Escherichia coli. Int J Biochem Cell Biol 38: 1050-1062
Missiakas D, Georgopoulos C, Raina S (1993) Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo. Proc Natl Acad Sci USA 90: 7084-7088
Mitobe J, Arakawa E, Watanabe H (2005) A sensor of the two-component system CpxA affects expression of the type III secretion system through posttranscriptional processing of InvE. J Bacteriol 187: 107-113
Moro EM, Weiss RD, Friedrich RS, de Vargas AC, Weiss LH, Nunes MP (1999) Aeromonas hydrophila isolated from cases of bovine seminal vesiculitis in south Brazil. J Vet Diagn Invest 11: 189-191
Pepe CM, Eklund MW, Strom MS (1996) Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion. Mol Microbiol 19: 857-869
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: 1169-1182
Raivio TL, Popkin DL, Silhavy TJ (1999) The Cpx envelope stress response is controlled by amplification and feedback inhibition. J Bacteriol 181: 5263-5272
Rietsch A, Bessette P, Georgiou G, Beckwith J (1997) Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin. J Bacteriol 179: 6602-6608
Rodrigue A, Quentin Y, Lazdunski A, Mejean V, Foglino M (2000) Two-component systems in Pseudomonas aeruginosa: why so many? Trends Microbiol 8: 498-504
Rodriguez LA, Ellis AE, Nieto TP (1992) Purification and characterisation of an extracellular metalloprotease, serine protease and haemolysin of Aeromonas hydrophila strain B32: all are lethal for fish. Microb Pathog 13: 17-24
Sechi LA, Deriu A, Falchi MP, Fadda G, Zanetti S (2002) Distribution of virulence genes in Aeromonas spp. isolated from Sardinian waters and from patients with diarrhoea. J Appl Microbiol 92: 221-227
Sha J, Pillai L, Fadl AA, Galindo CL, Erova TE, Chopra AK (2005) The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila. Infect Immun 73: 6446-6457
Shevchik VE, Condemine G, Robert-Baudouy J (1994) Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity. EMBO J 13: 2007-2012
Stafford SJ, Humphreys DP, Lund PA (1999) Mutations in dsbA and dsbB, but not dsbC, lead to an enhanced sensitivity of Escherichia coli to Hg2+ and Cd2+. FEMS Microbiol Lett 174: 179-184
Stewart EJ, Aslund F, Beckwith J (1998) Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. Embo J 17: 5543-5550
Sun XX, Wang CC (2000) The N-terminal sequence (residues 1-65) is essential for dimerization, activities, and peptide binding of Escherichia coli DsbC. J Biol Chem 275: 22743-22749
Vinci F, Couprie J, Pucci P, Quemeneur E, Moutiez M (2002) Description of the topographical changes associated to the different stages of the DsbA catalytic cycle. Protein Sci 11: 1600-1612
Vipond R, Bricknell IR, Durant E, Bowden TJ, Ellis AE, Smith M, MacIntyre S (1998) Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida. Infect Immun 66: 1990-1998
Voss LM, Rhodes KH, Johnson KA (1992) Musculoskeletal and soft tissue Aeromonas infection: an environmental disease. Mayo Clin Proc 67: 422-427
Wong CY, Heuzenroeder MW, Flower RL (1998) Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model. Microbiology 144: 291-298
Yu HB, Kaur R, Lim S, Wang XH, Leung KY (2007) Characterization of extracellular proteins produced by Aeromonas hydrophila AH-1. Proteomics 7: 436-449
Yu HB, Zhang YL, Lau YL, Yao F, Vilches S, Merino S, Tomas JM, Howard SP, Leung KY (2005) Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol 71: 4469-4477
Zhang YL, Lau YL, Arakawa E, Leung KY (2003) Detection and genetic analysis of group II capsules in Aeromonas hydrophila. Microbiology 149: 1051-1060