Welcome to International Network for Natural Sciences | INNSpub

Molecular characterization of Vibrio harveyi virulence-associated serine protease and outer membrane protein genes for vaccine development

Research Paper | March 1, 2016

| Download 6

Aslizah Mohd Aris, Ina-Salwany Md Yasin, Mohd Zamri-Saad, Hassan Mohd Daud, Norfarrah Mohamed Alipiah

Key Words:

Int. J. Biosci.8( 3), 10-28, March 2016

DOI: http://dx.doi.org/10.12692/ijb/8.3.10-28


IJB 2016 [Generate Certificate]


Vibrio harveyi is the most pathogenic species associated with the infection in a wide range of marine species in aquaculture industries. A few virulence-associated genes have been discovered in V. harveyi. This study reports the cloning, sequence analysis and phylogenetic study of serine protease (VHS) and outer membrane protein (OMP) from a pathogenic V. harveyi, which isolated from a local outbreak of diseased tiger grouper. Molecular identification revealed that VHS and OMP consist of 1368 and 816 base pairs and encoded for mature peptides of 429 and 251 amino acids, respectively. The amino acids sequence identities of VHS was 100% similarity with protease of V. harveyi and OMP was 99% of membrane protein of V. harveyi, as compared to published sequence. Phylogenetic analysis and conserved domain search proposed that VHS is a serine endoprotease DegQ and OMP is an OmpK type. Signal peptide, transmembrane -barrel and subcellular localization have supported the findings whereby demonstrated VHS belonged to periplasmic serine protease DegQ, composed one -barrel and two -barrels region. OMP displayed six -barrels and twelve -barrels suggesting it is belonged to outer membrane integral membrane protein, specifically act as a porin type outer membrane protein. Prediction of antigenic sites revealed that VHS composed 62 sites and OMP have 36 antigenic sites, assuming that they can provoke immune response of the infected hosts. In conclusion, it is strongly suggests that both genes can be potentially used for developing an effective live-attenuated vaccine candidate against vibriosis and further be applied in aquaculture industries.


Copyright © 2016
By Authors and International Network for
Natural Sciences (INNSPUB)
This article is published under the terms of the Creative
Commons Attribution Liscense 4.0

Molecular characterization of Vibrio harveyi virulence-associated serine protease and outer membrane protein genes for vaccine development

Austin B, Zhang XH. 2006. V. harveyi: a significant pathogen of marine vetebrates and invertebrates.  Letters  in  Applied  Microbiology  43, 119-124. http://dx.doi.org/10.1111/j.1472-765X.2006.01989.x

Bagos PG, Liakopoulos TD, Spyropoulos IC, Hamodrakas SJ. 2004. PRED-TMBB: A web server for predicting the topology of b-barrel outer membrane proteins. Nucleic Acids Research 32, W400-W404. http://dx.doi.org/10.1093/nar/gkh417

Bannwarth M, Schulz GE. 2003. The expression of outer membrane proteins for crystallization. Biochimica et Biophysica Acta 1610, 37-45. http://dx.doi.org/10.1016/S0005-2736(02)00711-3

Cheng S, Zhang WW, Zhang M, Sun L. 2010. Evaluation  of  the  vaccine  potential  of  a  cytotoxic protease and a protective immunogen from a pathogenic V. harveyi strain. Vaccine 28(4), 1041-1047. http://dx.doi.org/10.1016/j.vaccine.2009.10.122

Chou KC, Elrod DW. 1999. Prediction of membrane protein types and subcellular locations. Proteins 34, 137-153. http://dx.doi.org/10.1002/(SICI)10970134(19990101)34:1<137::AID-PROT11>3.0.CO;2-O

Defoirdt T. 2014. Virulence mechanisms of bacterial aquaculture pathogens and antivirulence therapy for aquaculture. Reviews in Aquaculture 6(2), 100-114. http://dx.doi.org/10.1111/raq.12030

Finlay BB, Falkow S. 1997. Common themes in microbial pathogenicity revisited. Microbiology and Molecular Biology Reviews 61, 136-169

Food and Agriculture Organization of the United Nations (FAO). 2014. World Review of Fisheries and Aquaculture Topic: Status and Trends. In: The State of World Fisheries and Aquaculture 2014: Opportunities and challenges, Rome, 223 pp. http://www.fao.org/3/a-i3720e.pdf

Frey J. 2007. Biological safety concepts of genetically modified live bacterial vaccines. Vaccine 25(30), 5598-5605. http://dx.doi.org/10.1016/j.vaccine.2006.11.058

Huang X, Madan A. 1999. CAP3: A DNA sequence assembly program. Genome Research 9, 868-877. http://dx.doi.org/10.1101/gr.9.9.868

Inoue T, Matsuzaki S, Tanaka S. 1995. Cloning and  sequence  analysis  of  Vibrio parahaemolyticusomp Kgene encoding a 26-kDa outer membrane protein, OmpK, that serves as receptor for a broad-host-range vibriophage, KVP40. FEMS Microbiology Letters 134, 245-249. http://dx.doi.org/10.1111/j.15746968.1995.tb07945.x

Kawai K, Liu Y, Ohnishi K, Oshima S. 2004. A conserved 37 kDa outer membrane protein of Edwardsiella tarda is an effective vaccine candidate. Vaccine 22(25-26), 3411–3418. http://dx.doi.org/10.1016/j.vaccine.2004.02.026

Kazemian HB, Yusuf SA, White K. 2014. Signal peptide discrimination and cleavage site identification using SVM and NN. Computers in Biology and Medicine 45, 98-110. http://dx.doi.org/10.1016/j.compbiomed.2013.11.017

Li N, Yang Z, Bai J, Fu X, Liu L, Shi C, Wu S. 2010. A shared antigen among Vibrio species: Outer membrane protein-OmpK as a versatile Vibriosis vaccine candidate in Orange-spotted grouper (Epinephelus coioides). Fish and Shellfish Immunology 28(5-6), 952-956. http://dx.doi.org/10.1016/j.fsi.2010.02.010

Li ZH, Tang ZX, Fang XJ, Zhang ZL, Shi LE. 2013. Bioinformatics analysis of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica. Computational Biology and Chemistry 47, 207-214. http://dx.doi.org/10.1016/j.compbiolchem.2013.09.0 03

Lutwyche P, Exner MM, Hancock REW, Trust TJ. 1995. A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout. Infection and Immunity 63(8), 3137-3142.

Ma Y, Zhang Y, Zhao D. 2008. Polyvalent attenuated live vaccine for preventing and curing vibriosis of cultivated fish. Patent No: US2008/0274136 A1.

Mao Z, Yu L, You Z, Wei Y, Liu Y. 2007. Cloning, expression and immunogenicity analysis of five outer membrane  proteins  of  Vibrio  parahaemolyticus  zj 2003. Fish and Shellfish Immunology 23(3), 567-575. http://dx.doi.org/10.1016/j.fsi.2007.01.004

Mims CA, Nash A, Stephen J. 2001. Vaccines and How They Work, In: Mims’ Pathogenesis of Infectious Disease (Fifth Edition), USA, Academic Press.392-415.

Nancy BS, Owens L. 2013. Virulence changes to Harveyi clade bacteria infected with bacteriophage from Vibrio owensii Indian Journal of Virology 24(2), 180-187. http://dx.doi.org/10.1007/s13337-013-0136-1

Nehlah Rosli, Ina-Salwany Md Yasin, Murni Karim, Nur-Nazifah, Siti Zaharah Abdullah. Molecular characterization and antigenicity of outer membrane protein (OMP) of Vibrio alginolyticus, isolated from diseased Tiger Grouper (Epinephelus fuscoguttatus). In Proceeding of the 9th Symposium on Diseases in Asian Aquaculture (DAA9), 24 – 28 November 2014, Ho Chi Minh City, Vietnam.

Nielsen H, Engelbrecht J, Brunak S, Von-Heijne G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10(1), 1-6. http://dx.doi.org/10.1093/protein/10.1.1

Nikaido H. 2003. Molecular basis of bacterial outer membrane permeability revisited. Microbiology and Molecular Biology Reviews 67(4), 593-656. http://dx.doi.org/10.1128/MMBR.67.4.593-656.2003

Ningqiu L, Junjie B, Shuqin W, Xiaozhe F, Haihua L, Xing Y, Cunbin S. 2008. An outer membrane protein, OmpK is an effective vaccine candidate for V. harveyi in Orange-spotted grouper (Epinephelus coioides). Fish and Shellfish Immunology 25(6), 829-833. http://dx.doi.org/10.1016/j.fsi.2008.09.007

Petersen TN, Brunak S, Von-Heijne G, Nielsen H. 2011. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nature Methods 8(10), 785-786. http://dx.doi.org/10.1038/nmeth.1701

Plant KP, LaPatra SE. 2011. Advances in fish vaccine delivery. Developmental and Comparative Immunology 35(12), 1256-1262. http://dx.doi.org/10.1016/j.dci.2011.03.007

Polgár L. 2005. Review: The catalytic triad of serinepeptidases. Cellular and Molecular Life Sciences 62(19), 2161-2172. http://dx.doi.org/10.1007/s00018-005-5160-x

Qin YX, Yan QP. The antigenicity of the flagellin, outer membrane proteins and lipopolysaccharide of Vibrio harveyi. In Bioinformatics and Biomedical Technology (ICBBT), 2010 International Conference on Bioinformatics and Biomedical Technology. 16-18 April 2010. Chengdu, China. 292-295 p. http://dx.doi.org/10.1109/ICBBT.2010.5478956

Rahman MH, Kawai K. 2000. Outer membrane proteins of Aeromonas hydrophila induce protective immunity in goldfish. Fish and Shellfish Immunology 10(4), 379-382. http://dx.doi.org/10.1006/fsim.1999.0245

Rawlings ND, Barret AJ, Bateman A. 2010. MEROPS: The peptidase database. Nucleic Acids Research 38, D227-233. http://dx.doi.org/10.1093/nar/gkp971

Sarjito Radjasa OK, Sabdono A, Prayitno SB, Hutabarat S. 2009. Phylogenetic diversity of the causative agents of Vibriosis associated with groupers fish from Karimunjawa Islands, Indonesia. Current Research in Bacteriology 2(1), 14-21. http://dx.doi.org/10.3923/crb.2009.14.21

Sawabe T, Ogura Y, Matsumura Y, Feng G, Rohul Amin AKM, Mino S, Nakagawa S, Sawabe T, Kumar R, Fukui Y, Satomi M, Matsushima R, Thompson FL, Gomez-Gil B, Christen R, Maruyama F, Kurokawa K, Hayashi T. 2013. Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Frontiers in Microbiology 4(414), 1-14. http://dx.doi.org/10.3389/fmicb.2013.00414

Schulz GE. 2002. The structure of bacterial outer membrane proteins. Biochimica et Biophysica Acta 1565(2), 308-317. http://dx.doi.org/10.1016/S0005-2736(02)00577-1

Shoemaker CA, Klesius PH, Evans JJ, Arias CR. 2009. Use of modified live vaccines in aquaculture. Journal of the World Aquaculture Society. 40(5), 573-585. http://dx.doi.org/10.1111/j.1749-7345.2009.00279.x

Spiess C, Beil A, Ehrmann M. 1999. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 339-347. http://dx.doi.org/10.1016/S0092-8674(00)80743-6

Sun  K,  Zhang  WW,  Hou  JH,  Sun  L.  2009. Immunoprotective analysis of VhhP2, a Vibrio harveyi  vaccine  candidate.  Vaccine  27(21), 2733-2740. http://dx.doi.org/10.1016/j.vaccine.2009.03.012

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30(12), 2725-2729. http://dx.doi.org/10.1093/molbev/mst197

Vanmalae S, Defoirdt T, Cleenwerck I, De Vos P, Bossier P. 2015. Characterization of the virulence of Harveyi clade Vibrios isolated from a shrimp hatchery in vitro and in vivo, in a brine shrimp (Artemia franciscana) model system. Aquaculture 435, 28-32. http://dx.doi.org/10.1016/j.aquaculture.2014.09.015

Vázquez-Juárez RC, Barrera-Saldaña HA, Hernández-Saavedra NY, Gómez Chiarri M, Ascencio F. 2003. Molecular cloning, sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii. Journal of Applied Microbiology 94(5), 908-918. http://dx.doi.org/10.1046/j.1365-2672.2003.01928.x

Waller PRH, Sauer RT. 1996. Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease. Journal of Bacteriology 178(4), 1146-1153.

Won KM, Park SI. 2008. Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea. Aquaculture 285(1-4), 8-13. http://dx.doi.org/10.1016/j.aquaculture.2008.08.013

Ye J, van den Berg B. 2004. Crystal structure of the bacterial nucleoside transporter Tsx. The EMBO Journal 23(16), 3187-3195. http://dx.doi.org/10.1038/sj.emboj.7600330

Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FSL. 2010. PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26(13), 1608-1615. http://dx.doi.org/10.1093/bioinformatics/btq249

Zhang W-W, Sun K, Cheng S, Sun L. 2008. Characterization of DegQVh, a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain. Applied and Environmental Microbiology 74(20), 6254-6262. http://dx.doi.org/10.1128/AEM.00109-08

Zhang C, Yu L, Qian R. 2007. Characterization of OmpK, GAPDH and their fusion OmpK-GAPDH derived from Vibrio harveyi outer membrane proteins: Their immunoprotective ability against vibriosis in large yellow croaker (Pseudosciaena crocea).  Journal  of  Applied  Microbiology  103(5), 1587-1599. http://dx.doi.org/10.1111/j.1365-2672.2007.03386.x

Zhang X, Robertson P, Austin B, Xu H. 1997. Comparison of outer membrane protein profiles of Vibrio sp. Acta Microbiologica Sinica 37(6), 449-454.

Zhao Y, Liu Q, Wang X, Zhou L, Wang Q, Zhang Y. 2011. Surface display of Aeromonas hydrophila GAPDH in attenuated Vibrio anguillarum to develop a noval multivalent vector vaccine. Marine Biotechnology 13(5), 963-970. http://dx.doi.org/10.1007/s10126-010-9359-y

Zheng Z, Xiao Y, Wu H, Wang Q, Xiao J, Zhang Y, Liu Q. 2012. Different approaches to expressing Edwardsiella tarda antigen GAPDH in attenuated Vibrio anguillarum for multivalent fish vaccines. Journal of Fish Diseases 35, 569-577. http://dx.doi.org/10.1111/j.1365-2761.2012.01381.x

Zhou Z, Pang H, Ding Y, Cai J, Huang Y, Jian J, Wu Z. 2013. VscO, a putative T3SS chaperone escort of Vibrio alginolyticus, contributes to virulence in fish and is a target for vaccine development. Fish and Shellfish Immunology 35(5), 1523-1531. http://dx.doi.org/10.1016/j.fsi.2013.08.017

Zou L, Wang Z, Wang Y, Hu F. 2010. Combined prediction of transmembrane topology and signal peptide of b-barrel proteins: Using a hidden Markov model and genetic algorithms. Computers in Biology and Medicine 40(7), 621-628. http://dx.doi.org/10.1016/j.compbiomed.2010.04.00 6