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dc.contributor.authorNúñez-Díaz, José Alberto
dc.contributor.authorFumanal, Milena
dc.contributor.authorGarcía de la Banda, Inés
dc.contributor.authorMoriñigo-Gutierrez, Miguel Angel 
dc.contributor.authorBalebona-Accino, Maria del Carmen 
dc.date.accessioned2016-09-27T11:10:31Z
dc.date.available2016-09-27T11:10:31Z
dc.date.created2016
dc.date.issued2016-09-27
dc.identifier.urihttp://hdl.handle.net/10630/12100
dc.description.abstractThe marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting several fish species including flatfish Solea senegalensis (Kaup). Phdp is able to avoid host defences by invasion and intracellular survival in non-phagocytic cells, mainly epithelial cells. Virulence factors reported in Phdp include restricting complement-mediated activity, apoptosis of phagocytes caused by exotoxins secretion, iron acquisition mechanisms such as siderophores that enable the pathogen to obtain iron from transferrin and ability to bind haemin and antioxidant enzymatic activities capable to counteract superoxide radicals (Do Vale et al., 2005; Andreoni and Magnano, 2014). Commonly, genes expressed during pathogen infection are important for pathogenicity. In vivo-induced antigen technology (IVIAT) (Handfield et al., 2000) has been used to identify in vivo-induced genes using pooled sera from fish that have experienced photobacteriosis. Materials and methods Sera were obtained from surviving S. senegalensis specimens after sublethal infection with Phdp (Lg41/01) and subsequently pooled and adsorbed against in vitro grown Phdp Lg41/01 and Escherichia coli BL21 (DE3) cells and lysates according to Handfield et al. (2000). The efficiency of sera adsorption was evaluated based on the immunoreactivity after each adsorption step with whole and lysed Phdp cells grown in vitro. A genomic expression library of Phdp Lg41/01 was generated in E. coli BL21 (DE3) using pET-30 expression system (Novagen, San Diego, CA, USA). The expression library was probed with adsorbed and non-absorbed sera using immunoblot technique. Reactive clones of in vivo-induced and in vitro antigens were obtained, purified and their inserted DNA sequenced (Macrogen Europe, Amsterdam, The Netherlands). Nucleotide sequences were compared against the NCBI protein database using BLASTx. Results A progressive reduction in sera immunoreactivity against in vitro grown Phdp cells was detected after the adsorption rounds, especially after the first adsorption step. Thus, following adsorption steps substantially removed antibodies against in vitro expressed antigens and resulted in relative enrichment in antibodies recognizing in vivo expressed antigens. The library from Phdp Lg14/01 constructed in E. coli BL21 (DE3) consisted of approximately 6500 recombinants. A total of 117 clones were selected for their reactivity with pooled adsorbed and non-adsorbed sera from convalescent S. senegalensis specimens after a first round of screening. In a second screening, 14 out of 117 candidate clones showed positive reaction, among which two clones were clearly positive and two gave weak reaction against adsorbed sera. Predicted proteins codified by inserted sequences have intracellular and membrane cell location and are involved in virulence, synthesis of intermediary products, energy metabolism and gene replication. Inosine-5’-monophosphate dehydrogenase (IMPDH) and alkyl hydroperoxide reductase (AhpC) have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp. Iron/manganese superoxide dismutase (Fe/Mn-SOD) and alanyl-tRNA synthetase (AlaRS) proteins have also been identified, though with weak signal. Discussion and conclusion Identification of immunogenic bacterial proteins during Phdp infection is essential for understanding bacterial pathogenesis and development of effective vaccines. AhpC peroxidase activity has a protective role by reducing hydrogen peroxide, peroxynitrite and organic hydroperoxides. Immunization with AhpC conferred protection against Helicobacter pylori infection (O’Riordan et al., 2012). IMPDH catalyzes the conversion of products essential in de novo synthesis of guanine nucleotides. Adequate levels of purine nucleotides are critical for cell proliferation, nucleic acid replication, cell signaling and as a biochemical energy source. This gene is an important therapeutic target against bacterial diseases (Shu and Nair,2008). In conclusion, different genes expressed during Phdp infection in S. senegalensis have been identified. Among them, IMPDH and AhpC have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp. Thus, they are likely to play a role in the virulence of Phdp. The antigenic character of these proteins makes them potential targets for the development of new vaccines. References Andreoni, F., and Magnani, M., 2014. Photobacteriosis: Prevention and Diagnosis. Journal of Immunology Research, 2014: 1-7. Do Vale, A., Silva, M.T., dos Santos, N.M., Nascimento, D.S., Reis Rodrigues, P., Costa Ramos, C., Ellis, A.E., and Azevedo, J.E., 2005. AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils. Molecular Microbiology, 58: 1025-1038. Handfield, M., Brady, L.J., Progulske-Fox, A., and Hillman, J.D., 2000. IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends in Microbiology, 8: 336-339. O’Riordan A.A., Morales V.A., Mulligan L., Faheem N., Windle H.J., and Kelleher D.P., 2012. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine. Vaccine, 30:3876-3884. Shu, Q., and Nair, V., 2008. Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery. Medicinal Research Reviews, 28:219-232.es_ES
dc.description.sponsorshipUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.es_ES
dc.language.isoenges_ES
dc.rightsinfo:eu-repo/semantics/openAccesses_ES
dc.subjectBacterias patógenases_ES
dc.subject.otherPhotobacteriumes_ES
dc.subject.otherSoleaes_ES
dc.subject.otherIVIATes_ES
dc.titleUse of in-vivo induced antigen technology to identify bacterial genes expressed during Solea senegalensis infection with Photobacterium damselae subsp. piscicidaes_ES
dc.typeinfo:eu-repo/semantics/conferenceObjectes_ES
dc.centroFacultad de Cienciases_ES
dc.relation.eventtitleAquaculture Europe 16. Food for thoughtses_ES
dc.relation.eventplaceEdimburgo, Reino Unidoes_ES
dc.relation.eventdateseptiembre 2016es_ES
dc.identifier.orcidhttp://orcid.org/0000-0001-7421-2792es_ES
dc.cclicenseby-nc-ndes_ES


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