- Email: [email protected]
Metalloproteinase activity in the hemolymph of hydrothermal vent mussel Bathymodiolus azoricus
1666
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
environmentally sensitive. To evaluate this possibility we carried out structure/function studies in neutral and acid environments by means of biophysical approaches. Infrared spectroscopic data indicate that the structure of Myt C is pH dependent: acid media increases the content of alpha helix and beta-sheet and reduces random structure. Furthermore, at low pH, Myt C was able to efficiently aggregate artificial phospholipid membranes suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial activity. If biological approaches confirmed these biophysical results, the pH-dependent properties of Myt C may allow the design of agents that would function selectively in specific pH environments such as the gastric lumen. * Corresponding author. E-mail address: [email protected] (A. Estepa) xThese authors have contributed equally to this work.
O-450. Metalloproteinase activity in the hemolymph of hydrothermal vent mussel Bathymodiolus azoricus I. Martins 1, *, R. Company 2, T. Cerqueira 1, M.J. Bebianno 2, R.S. Santos 1, R. Bettencourt 1. 1 IMAR, DOP – Department of Oceanography and Fisheries, University of the Azores, Horta, Portugal; 2 CIMA, Faculty of Science and Technology, University of Algarve, Faro, Portugal
Abstract The extreme conditions present at the hydrothermal vent ecosystems such as, high temperature and pressure, high concentrations of trace metals, toxic gases such as methane (CH4), carbon dioxide (CO2) and hydrogen sulfide (H2S) could apparently be deleterious to the aerobic organisms. However, B. azoricus developed physiological strategies to cope with such inhospitable environment being the most successful and widespread species in the Mid Atlantic Ridge hydrothermal vents. Such remarkable adaptive response to environmental stressors must represent readjustments on normal biochemical reactions in order to maintain the integrity of cell function and metabolism. In bivalve molluscs, hemolymph and the circulating cells hemocytes, forms a primary line of defense against infectious agents and cellular stressful factors. Therefore, metalloproteinase expression in B. azoricus hemolymph was used as a molecular indicator of the environmental effect on mussel physiology. Individuals collected at Menez Gwen hydrothermal vent field were acclimatized at atmospheric pressure during 1 month under different experimental conditions i) iron sulfate [Fe2(SO4)3] exposure; ii) CH4 and H2S supply; iii) plain sea water. With substrate gel zymography we found that samples of both fresh collected and acclimatized mussel's strongly express proteolytic activity, especially after 1 week exposure to iron sulfate and plain sea water acclimatization. In parallel the 2-D SDS-PAGE technique allowed the analysis of the protein expression profiles that match the proteolytic activity found. This will reveal new biomarkers of proteomic signatures in B. azoricus. The present study brings new insights into vent mussel cellular control and defense mechanisms behind the physiological strategies to overcome such extreme ecosystem. * Corresponding author. E-mail address: [email protected] (I. Martins)
O-433. The role of apoptosis in immunomodulation by beta-glucan during in vitro viral infection J.J. Miest 1, *, M. Adamek 2, D. Steinhagen 2, D. Hoole 1. 1
School of Life Sciences, Keele University, Keele, UK; Fish Disease Research Unit, Centre for Infection Medicine, University of Veterinary Medicine in Hanover, Hanover, Germany 2
Abstract The use of immunomodulating substances such as b-glucan has been shown to increase survival during viral infections in fish. Various underlying mechanisms to explain this effect have been proposed, however the potential role of apoptosis, i.e. a form of programmed cell death, which is an important factor in the immune response against viral infections, has been neglected. The anti-viral response often includes the induction of apoptosis in infected cells in order to limit the reproduction and distribution of the virus. The influence of b-glucan on the apoptotic process is largely unknown, although it can influence apoptosis-related gene expression following exposure to LPS and Poly(I:C). Hence we aimed to ascertain the effects of immunomodulation on the apoptotic process during viral infections to understand if apoptosis plays a role in disease protection. We used spring viremia of carp virus (SVCV) and Cyprinid herpesvirus 3 (CyHV-3) in an in vitro model, as they cause extensive mortalities in aquaculture during disease outbreaks, and bglucan, in the form of the feed supplement MacroGardÒ, as an immunomodulator. Levels of apoptosis were assessed through microscopy using acridine orange staining, and genetic analysis targeting the proapoptotic genes p53, caspase 9, apaf-1 and the anti-apoptotic gene IAP, as well as the nitric oxide producing enzyme iNOS. The results indicated that the effect of the immunomodulant on apoptosis varied depending on the infectious agent. During the SVCV infection, viral-induced apoptosis and some apoptosis related genes seemed to be enhanced in the infected cell cultures by previous exposure to MacroGardÒ. However, no such effect was observed with CyHV-3 infection. These results could explain some of the protective effects of b-glucan against viral infections, but also suggest the role of immunomodulants on apoptosis may not be consistent across all disease systems. We will discuss these findings in the context of our recent research exploring virus-induced apoptosis. The research leading to these results has received funding from the European Community's Seventh Framework Programme ([FP7/2007-2013] under grant agreement n PITN-GA-2008-214505). * Corresponding author. E-mail address: [email protected] (J.J. Miest)
O-394. Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray R. Moreira 1, M. Milan 2, P. Balseiro 1, A. Romero 1, M. Babbucci 2, A. Figueras 1, L. Bargelloni 2, B. Novoa 1, *. 1
Instituto de Investigaciones Marinas, IIM – CSIC, Eduardo Cabello, 6, 362018 Vigo, Spain; 2 Department of Comparative Biomedicine and Food Science (BCA) University of Padova, Viale dell'Università 16, 35020 Legnaro, Italy Abstract Background: The Manila clam (Ruditapes philippinarum) is a cultured bivalve with worldwide commercial importance, and diseases in this species can cause high economic losses. For this reason, interest in the immune genes in bivalves has recently increased. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and the application of this microarray to study the gene transcription profiles of hemocytes from clams that were challenged with V. alginolyticus through a time course. Results: The complete set of sequences from R. philippinarum available in the public databases and the hemocyte sequences enriched in immune transcripts (Moreira et al., 2012) were assembled successfully. A total of 12,156 annotated target sequences were used to construct the 8x15k oligomicroarray. The microarray experiments and analysis yielded a total of 579 differentially expressed transcripts. Using the gene expression results, the associated Gene Ontology terms and the enrichment analysis, we found different response mechanisms throughout the infection. Genes related to signaling, transcription and apoptosis, such as IL-17D, NF-kB or calmodulin, were typically expressed as early as 3 h post-infection (hpi), while characteristic immune genes, such as PGRPs, FREPs and defense proteins
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
environmentally sensitive. To evaluate this possibility we carried out structure/function studies in neutral and acid environments by means of biophysical approaches. Infrared spectroscopic data indicate that the structure of Myt C is pH dependent: acid media increases the content of alpha helix and beta-sheet and reduces random structure. Furthermore, at low pH, Myt C was able to efficiently aggregate artificial phospholipid membranes suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial activity. If biological approaches confirmed these biophysical results, the pH-dependent properties of Myt C may allow the design of agents that would function selectively in specific pH environments such as the gastric lumen. * Corresponding author. E-mail address: [email protected] (A. Estepa) xThese authors have contributed equally to this work.
O-450. Metalloproteinase activity in the hemolymph of hydrothermal vent mussel Bathymodiolus azoricus I. Martins 1, *, R. Company 2, T. Cerqueira 1, M.J. Bebianno 2, R.S. Santos 1, R. Bettencourt 1. 1 IMAR, DOP – Department of Oceanography and Fisheries, University of the Azores, Horta, Portugal; 2 CIMA, Faculty of Science and Technology, University of Algarve, Faro, Portugal
Abstract The extreme conditions present at the hydrothermal vent ecosystems such as, high temperature and pressure, high concentrations of trace metals, toxic gases such as methane (CH4), carbon dioxide (CO2) and hydrogen sulfide (H2S) could apparently be deleterious to the aerobic organisms. However, B. azoricus developed physiological strategies to cope with such inhospitable environment being the most successful and widespread species in the Mid Atlantic Ridge hydrothermal vents. Such remarkable adaptive response to environmental stressors must represent readjustments on normal biochemical reactions in order to maintain the integrity of cell function and metabolism. In bivalve molluscs, hemolymph and the circulating cells hemocytes, forms a primary line of defense against infectious agents and cellular stressful factors. Therefore, metalloproteinase expression in B. azoricus hemolymph was used as a molecular indicator of the environmental effect on mussel physiology. Individuals collected at Menez Gwen hydrothermal vent field were acclimatized at atmospheric pressure during 1 month under different experimental conditions i) iron sulfate [Fe2(SO4)3] exposure; ii) CH4 and H2S supply; iii) plain sea water. With substrate gel zymography we found that samples of both fresh collected and acclimatized mussel's strongly express proteolytic activity, especially after 1 week exposure to iron sulfate and plain sea water acclimatization. In parallel the 2-D SDS-PAGE technique allowed the analysis of the protein expression profiles that match the proteolytic activity found. This will reveal new biomarkers of proteomic signatures in B. azoricus. The present study brings new insights into vent mussel cellular control and defense mechanisms behind the physiological strategies to overcome such extreme ecosystem. * Corresponding author. E-mail address: [email protected] (I. Martins)
O-433. The role of apoptosis in immunomodulation by beta-glucan during in vitro viral infection J.J. Miest 1, *, M. Adamek 2, D. Steinhagen 2, D. Hoole 1. 1
School of Life Sciences, Keele University, Keele, UK; Fish Disease Research Unit, Centre for Infection Medicine, University of Veterinary Medicine in Hanover, Hanover, Germany 2
Abstract The use of immunomodulating substances such as b-glucan has been shown to increase survival during viral infections in fish. Various underlying mechanisms to explain this effect have been proposed, however the potential role of apoptosis, i.e. a form of programmed cell death, which is an important factor in the immune response against viral infections, has been neglected. The anti-viral response often includes the induction of apoptosis in infected cells in order to limit the reproduction and distribution of the virus. The influence of b-glucan on the apoptotic process is largely unknown, although it can influence apoptosis-related gene expression following exposure to LPS and Poly(I:C). Hence we aimed to ascertain the effects of immunomodulation on the apoptotic process during viral infections to understand if apoptosis plays a role in disease protection. We used spring viremia of carp virus (SVCV) and Cyprinid herpesvirus 3 (CyHV-3) in an in vitro model, as they cause extensive mortalities in aquaculture during disease outbreaks, and bglucan, in the form of the feed supplement MacroGardÒ, as an immunomodulator. Levels of apoptosis were assessed through microscopy using acridine orange staining, and genetic analysis targeting the proapoptotic genes p53, caspase 9, apaf-1 and the anti-apoptotic gene IAP, as well as the nitric oxide producing enzyme iNOS. The results indicated that the effect of the immunomodulant on apoptosis varied depending on the infectious agent. During the SVCV infection, viral-induced apoptosis and some apoptosis related genes seemed to be enhanced in the infected cell cultures by previous exposure to MacroGardÒ. However, no such effect was observed with CyHV-3 infection. These results could explain some of the protective effects of b-glucan against viral infections, but also suggest the role of immunomodulants on apoptosis may not be consistent across all disease systems. We will discuss these findings in the context of our recent research exploring virus-induced apoptosis. The research leading to these results has received funding from the European Community's Seventh Framework Programme ([FP7/2007-2013] under grant agreement n PITN-GA-2008-214505). * Corresponding author. E-mail address: [email protected] (J.J. Miest)
O-394. Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray R. Moreira 1, M. Milan 2, P. Balseiro 1, A. Romero 1, M. Babbucci 2, A. Figueras 1, L. Bargelloni 2, B. Novoa 1, *. 1
Instituto de Investigaciones Marinas, IIM – CSIC, Eduardo Cabello, 6, 362018 Vigo, Spain; 2 Department of Comparative Biomedicine and Food Science (BCA) University of Padova, Viale dell'Università 16, 35020 Legnaro, Italy Abstract Background: The Manila clam (Ruditapes philippinarum) is a cultured bivalve with worldwide commercial importance, and diseases in this species can cause high economic losses. For this reason, interest in the immune genes in bivalves has recently increased. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and the application of this microarray to study the gene transcription profiles of hemocytes from clams that were challenged with V. alginolyticus through a time course. Results: The complete set of sequences from R. philippinarum available in the public databases and the hemocyte sequences enriched in immune transcripts (Moreira et al., 2012) were assembled successfully. A total of 12,156 annotated target sequences were used to construct the 8x15k oligomicroarray. The microarray experiments and analysis yielded a total of 579 differentially expressed transcripts. Using the gene expression results, the associated Gene Ontology terms and the enrichment analysis, we found different response mechanisms throughout the infection. Genes related to signaling, transcription and apoptosis, such as IL-17D, NF-kB or calmodulin, were typically expressed as early as 3 h post-infection (hpi), while characteristic immune genes, such as PGRPs, FREPs and defense proteins