RT Conference Proceedings
T1 Use of in-vivo induced antigen technology to identify bacterial genes expressed during Solea senegalensis infection with Photobacterium damselae subsp. piscicida
A1 Núñez-Díaz, José Alberto
A1 Fumanal, Milena
A1 García de la Banda, Inés
A1 Moriñigo-Gutiérrez, Miguel Ángel
A1 Balebona-Accino, María del Carmen
K1 Bacterias patógenas
AB The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important diseaseoutbreaks affecting several fish species including flatfish Solea senegalensis (Kaup). Phdp is able to avoid host defencesby invasion and intracellular survival in non-phagocytic cells, mainly epithelial cells. Virulence factors reported in Phdpinclude restricting complement-mediated activity, apoptosis of phagocytes caused by exotoxins secretion, iron acquisitionmechanisms such as siderophores that enable the pathogen to obtain iron from transferrin and ability to bind haemin andantioxidant 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 antigentechnology (IVIAT) (Handfield et al., 2000) has been used to identify in vivo-induced genes using pooled sera from fishthat have experienced photobacteriosis.Materials and methodsSera were obtained from surviving S. senegalensis specimens after sublethal infection with Phdp (Lg41/01) and subsequentlypooled and adsorbed against in vitro grown Phdp Lg41/01 and Escherichia coli BL21 (DE3) cells and lysates according toHandfield et al. (2000). The efficiency of sera adsorption was evaluated based on the immunoreactivity after each adsorptionstep 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 probedwith adsorbed and non-absorbed sera using immunoblot technique. Reactive clones of in vivo-induced and in vitro antigenswere obtained, purified and their inserted DNA sequenced (Macrogen Europe, Amsterdam, The Netherlands). Nucleotidesequences were compared against the NCBI protein database using BLASTx.ResultsA progressive reduction in sera immunoreactivity against in vitro grown Phdp cells was detected after the adsorptionrounds, especially after the first adsorption step. Thus, following adsorption steps substantially removed antibodies againstin vitro expressed antigens and resulted in relative enrichment in antibodies recognizing in vivo expressed antigens. Thelibrary 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 convalescentS. senegalensis specimens after a first round of screening. In a second screening, 14 out of 117 candidate clones showedpositive reaction, among which two clones were clearly positive and two gave weak reaction against adsorbed sera. Predictedproteins 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 duringS. 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 conclusionIdentification of immunogenic bacterial proteins during Phdp infection is essential for understanding bacterial pathogenesisand 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 pyloriinfection (O’Riordan et al., 2012). IMPDH catalyzes the conversion of products essential in de novo synthesis of guaninenucleotides. Adequate levels of purine nucleotides are critical for cell proliferation, nucleic acid replication, cell signalingand 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 potentialtargets for the development of new vaccines.ReferencesAndreoni, 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., andAzevedo, J.E., 2005. AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicidawith 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 genesexpressed 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 hydroperoxidereductase: 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. MedicinalResearch Reviews, 28:219-232.
YR 2016
FD 2016-09-27
LK http://hdl.handle.net/10630/12100
UL http://hdl.handle.net/10630/12100
LA eng
NO Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.
DS RIUMA. Repositorio Institucional de la Universidad de Málaga
RD 21 ene 2026