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Phenotypic characterization, virulence, and immunogenicity of Edwardsiella tarda LSE40 aroA mutant

  • Applied genetics and molecular biotechnology
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Abstract

Bacterial aro mutants are frequently used as live attenuated vaccines for domestic animals. In this study, we characterized Edwardsiella tarda strain LSE40 with a deletion in the aroA gene. In addition to autotrophy, the aroA mutant appeared to have delayed cell division and reductions in its swarming motility, biofilm formation, and production of translocator proteins in the type III secretory system. The mutant exhibited high virulence attenuation in turbot fish, Scophthalmus maximus (L.), where the 50 % lethal dose increased by more than 3 log10 via intraperitoneal (i.p.) injection and by >2 log10 via immersion exposure compared with the wild-type parent strain. A tissue persistence study showed that the mutant retained the ability to invade and spread in turbot and viable cells could be detected up to 28 days after i.p. infection and 21 days after immersion exposure. These results suggested a pleiotropic role for aroA in the physiological behavior of E. tarda. Turbot exhibited a good humoral response and the enhanced expression of innate immune factors, interleukin 1β and lysozyme, when vaccinated with aroA mutant at 105 CFU via i.p. injection and at 108 CFU via immersion exposure. However, the aroA mutant did not provide effective protection for turbot against edwardsiellosis following i.p. vaccination at doses of 104–106 CFU or immersion vaccination at doses of 106–108 CFU ml−1. We hypothesized that the aroA mutant did not trigger an appropriate T cell-immune response in turbot against infection of E. tarda.

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References

  • Barat S, Willer Y, Rizos K, Claudi B, Mazé A, Schemmer AK, Kirchhoff D, Schmidt A, Burton N, Bumann D (2012) Immunity to Intracellular Salmonella depends on surface-associated antigens. PLoS Pathog 8:e1002966. doi:10.1371/journal.ppat.1002966

    Article  CAS  Google Scholar 

  • Bar-Tana J, Howlett BJ, Hertz R (1980) Ubiquinone synthetic pathway in flagellation of Salmonella typhimurium. J Bacteriol 143:637–643

    CAS  Google Scholar 

  • Castro R, Bernard D, Lefranc MP, Sixc A, Benmansour A, Boudinot P (2011) T cell diversity and TcR repertoires in teleost fish. Fish Shellfish Immunol 31:644–654. doi:10.1016/j.fsi.2010.08.016

    Article  CAS  Google Scholar 

  • Choi SH, Kim KH (2011) Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus). Fish Shellfish Immunol 31:58–65. doi:10.1016/j.fsi.2011.03.006

    Article  CAS  Google Scholar 

  • Cook RA, Tappe JP (1985) Chronic enteritis associated with Edwardsiella tarda infection in Rockhopper penguins. J Am Vet Med Assoc 187:1219–1220

    CAS  Google Scholar 

  • Fletcher M (1977) The effects of culture concentration and age, time, and temperature on bacterial attachment to polystyrene. Can J Microbiol 23:1–6. doi:10.1139/m77-001

    Article  Google Scholar 

  • Hernanz Moral C, Flańo del Castillo E, López Fierro P, Villena Cortes A, Anguita Castillo J, Cascnó Soriano A, Sánchez Salazar M, Razquin Peralta B, Naharro Carrasco G (1998) Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live attenuated vaccine. Infect Immun 66:1813–1821

    CAS  Google Scholar 

  • Hertz R, Bar-Tana J (1977) Anaerobic electron transport in anaerobic flagellum formation in Escherichia coli. J Bacteriol 132:1034–1035

    CAS  Google Scholar 

  • Hirono I, Tange N, Aoki T (1997) Iron-regulated hemolysin gene from Edwardsiella tarda. Mol Microbiol 24:851–856. doi:10.1046/j.1365-2958.1997.3971760.x

    Article  CAS  Google Scholar 

  • Jobichen C, Chakraborty S, Li M, Zheng J, Joseph L, Mok YK, Leung KY, Sivaraman J (2010) Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda. PLoS One 5:e12910. doi:10.1371/journal.pone.0012910

    Article  Google Scholar 

  • Ling SHM, Wang XH, Xie L, Lim TM, Leung KY (2000) Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models. Microbiology 146:7–19

    CAS  Google Scholar 

  • Lisa AC, Ronald LT (1996) Development of a defined minimal medium for the growth of Edwardsiella ictaluri. Appl Environ Microbiol 62:848–852

    Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. doi:10.1006/meth.2001.1262

    Article  CAS  Google Scholar 

  • Malcova M, Karasova D, Rychlik I (2009) aroA and aroD mutations in influence biofilm formation in Salmonella enteritidis. FEMS Microbiol Lett 291:44–49. doi:10.1111/j.1574-6968.2008.01433.x

    Article  CAS  Google Scholar 

  • Marsden MJ, Vaughan LM, Foster TJ, Secombes CJ (1996) A live (delta aroA) Aeromonas salmonicida vaccine for furunculosis preferentially stimulates T-cell responses relative to B-cell responses in rainbow trout (Oncorhynchus mykiss). Infect Immun 64:3863–3869

    CAS  Google Scholar 

  • Mathew JA, Tan YP, Rao PSS, Lim TM, Leung KY (2001) Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity. Microbiology 147:449–457

    CAS  Google Scholar 

  • Mo ZL, Xiao P, Mao YX, Zou YX, Wang B, Li J, Xu YL, Zhang PJ (2007) Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.). Fish Shellfish Immunol 23:521–530. doi:10.1016/j.fsi.2006.11.00

    Article  Google Scholar 

  • Mohanty BR, Sahoo PK (2007) Edwardsiellosis in fish: a brief review. J Biosci 32:1331–1344. doi:10.1007/s12038-007-0143-8

    Article  CAS  Google Scholar 

  • O’Callaghan D, Maskell D, Liew FY, Easmon CS, Dougan G (1988) Characterization of aromatic- and purine-dependent Salmonella typhimurium: attenuation, persistence, and ability to induce protective immunity in BALB/c mice. Infect Immun 56:419–423

    Google Scholar 

  • Pittard AJ (1996) Biosythesis of aromatic amino acids. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium: cellular and molecular biology, 2nd edn. American Society for Microbiology, Washington, DC, pp 458–484

    Google Scholar 

  • Rao PSS, Lim TM, Leung KY (2001) Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates. Infect Immun 69:5689–5697. doi:10.1128/IAI.69.9.5689-5697.2001

    Article  CAS  Google Scholar 

  • Rao PSS, Lim TM, Leung KY (2003) Functional genomics approach to the identification of virulence genes involved in Edwardsiella tarda pathogenesis. Infect Immun 71:1343–1351. doi:10.1128/IAI.71.3.1343-1351.2003

    Article  CAS  Google Scholar 

  • Rao PSS, Yamada Y, Tan YP, Leung KY (2004) Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol Microbiol 53:573–586. doi:10.1111/j.1365-2958.2004.04123.x

    Article  CAS  Google Scholar 

  • Reed LJ, Muench H (1938) A simple method of estimating fifty percent end points. Am J Epidemiol 27:493–497

    Google Scholar 

  • Rege DV, Sreenivasan A (1954) The influence of folic acid and vitamin B12 on nucleic acid metabolism in microorganisms. J Biol Chem 210:373–380

    CAS  Google Scholar 

  • Rychlik I, Cardova L, Sevcik M, Barrow PA (2000) Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype. J Microbiol Methods 42:255–263. doi:10.1016/S0167-7012(00)00199-8

    Article  CAS  Google Scholar 

  • Sakai T, Kanai K, Osatomi K, Yoshikoshi K (2003) Identification of a 19.3-kDa protein in MRHA-positive Edwardsiella tarda: putative fimbrial major subunit. FEMS Microbiol Lett 226:127–133. doi:10.1016/S0378-1097(03)00608-6

    Article  CAS  Google Scholar 

  • Sakai T, Matsuyama T, Sano M, Iida T (2009) Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization. Microbiol Immunol 53:131–139. doi:10.1111/j.1348-0421.2009.00108.x

    Article  CAS  Google Scholar 

  • Sambrook JE, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, New York

    Google Scholar 

  • Sebkova A, Karasova D, Crhanova M, Budinska E, Rychlik I (2008) aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity. J Bacteriol 190:3155–3160. doi:10.1128/JB.00053-08

    Article  CAS  Google Scholar 

  • Shane B, Stokstad EL (1975) Transport and metabolism of folates by bacteria. J Biol Chem 25:2243–2245

    Google Scholar 

  • Tan YP, Zheng J, Tung SL, Rosenshine I, Leung KY (2005) Role of type III secretion in Edwardsiella tarda virulence. Microbiology 151:2301–2313. doi:10.1099/mic.0.28005-0

    Article  CAS  Google Scholar 

  • Thune RL, Fernandez DH, Batista J (1999) An aroA mutant of Edwardsiella ictaluri is safe and efficacious as live, attenuated vaccine. J Aquat Anim Health 11:358–372. doi:10.1577/1548-8667(1999)011<0358:AAMOEI>2.0.CO;2

    Article  Google Scholar 

  • Trebichavsky I, Splichalova A, Rychlik I, Hojna H, Muneta Y, Mori Y, Splichal I (2006) Attenuated aroA Salmonella enterica serovar typhimurium does not induce inflammatory response and early protection of gnotobiotic pigs against parental virulent LT2 strain. Vaccine 24:4285–4289. doi:10.1016/j.vaccine.2006.02.054

    Article  CAS  Google Scholar 

  • Vaughan LM, Smith PR, Foster TJ (1993) An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the salmonid disease furunculosis. Infect Immun 61:2172–2181

    CAS  Google Scholar 

  • Verstraeten N, Braeken K, Debkumari B, Fauvart M, Fransaer J, Vermant J, Michiels J (2008) Living on a surface: swarming and biofilm formation. Trends Microbiol 16:496–506. doi:10.1016/j.tim.2008.07.004

    Article  CAS  Google Scholar 

  • Wang B, Mo ZL, Mao YX, Zou YX, Xiao P, Li J, Yang JY, Ye XH, Leung KY, Zhang PJ (2009) Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC. Microbiology 155:1260–1271. doi:10.1099/mic.0.021865-0

    Article  CAS  Google Scholar 

  • Wang X, Wang Q, Xiao J, Liu Q, Wu H, Zhang Y (2010) Hemolysin EthA in Edwardsiella tarda is essential for fish invasion in vivo and in vitro and regulated by two-component system EsrA-EsrB and nucleoid protein HhaEt. Fish Shellfish Immunol 29:1082–1091. doi:10.1016/j.fsi.2010.08.025

    Article  CAS  Google Scholar 

  • Xiao P, Mo ZL, Zou YX, Wang B, Xu YL, Zhang PJ (2007) Immune responses and efficacy of an oral-emulsified bivalent vaccine against Vibrio anguillarum in turbot (Scophthalmus maximus). High Technol Lett 17:979–985

    Google Scholar 

  • Xiao J, Chen T, Wang Q, Liu Q, Wang X, Lv Y, Wu H, Zhang Y (2011) Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda. Lett Appl Microbiol 53:430–437. doi:10.1111/j.1472-765X.2011.03126.x

    Article  CAS  Google Scholar 

  • Xie HX, Yu HB, Zheng J, Nie P, Foster LJ, Mok YK, Finlay BB, Leung KY (2010) EseG, an effector of the type III secretion system of Edwardsiella tarda, triggers microtubule destabilization. Infect Immun 78:5011–5021. doi:10.1128/IAI.00152-10

    Article  CAS  Google Scholar 

  • Zhang M, Sun K, Sun L (2008) Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain. Microbiology 157:2060–2069. doi:10.1099/mic.0.2008/017343-0

    Article  Google Scholar 

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Acknowledgments

This project was supported by the National Natural Sciences Foundation of China (grant no. 31072245) and the planning project of “the twelfth five-year-plan” in the National Science and Technology for the Rural Development in China (2011BAD13B07) and in Marine Technology for Typical Marine Biological Development and Utilization of Important Functional Genes (2012AA092203).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao, 266071, People’s Republic of China

    Zhao-Lan Mo, Jie Li & Gui-Yang Li

  2. Key Lab of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071, People’s Republic of China

    Peng Xiao

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  1. Zhao-Lan Mo
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Correspondence to Zhao-Lan Mo.

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Mo, ZL., Li, J., Li, GY. et al. Phenotypic characterization, virulence, and immunogenicity of Edwardsiella tarda LSE40 aroA mutant. Appl Microbiol Biotechnol 97, 6325–6335 (2013). https://doi.org/10.1007/s00253-013-4813-1

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