Efficacy of heterologous and homologous heat shock protein 70s as protective agents to Artemia franciscana challenged with Vibrio campbellii Kartik Baruah, Jayant Ranjan, Patrick Sorgeloos, Peter Bossier * Laboratory of Aquaculture and Artemia Reference Center, Faculty of Bioscience Engineering, Ghent University, Rozier 44, 9000 Ghent, Belgium article info Article history: Received 10 April 2010 Received in revised form 28 June 2010 Accepted 2 July 2010 Available online 17 July 2010 Keywords: Heat shock protein (Hsp) 70 DnaK Phenoloxidase Artemia franciscana Vibrio campbellii abstract The Hsp70 class of heat shock proteins (Hsps) has been implicated at multiple points in the immune response of both vertebrates and invertebrates. This class of chaperones is highly conserved in both sequence and structure, from prokaryotes to higher eukaryotes. In view of their high degree of homology, it was assumed that these Hsp70 proteins derived either from the prokaryotes or eukaryotes would have similar functions, especially in relation to their protective ability in a challenge assay. To verify this, we compared two evolutionary diverse Hsp70s, Artemia Hsp70 and Escherichia coli Hsp70 equivalent DnaK (each overproduced in E.coli), for their ability to protect Artemia against Vibrio challenge. Results showed that Artemia fed with E. coli producing Artemia Hsp70 or DnaK proteins, as assessed by immune-probing in western blots, survived better in a Vibrio challenge assay. The observed effects could be due to enhancement of the Artemia immune system as phenoloxidase activity was found to be increased by these proteins. These two Hsp70 proteins exhibit a high degree of homology, particularly in the peptide- binding domain (the putative innate immunity-activating portion) with 59.6% identity, indicating that the observed protective capacity of homologous or heterologous Hsp70 proteins might reside within this peptide-binding domain. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Diseases caused by pathogenic or opportunistic bacteria, such as Vibrio spp. are still considered a major constraint to the sustainable development of aquaculture worldwide [1]. Although, the use of antibiotics and disinfectants had some success in the prevention or cure of such diseases [2], such practices are undesirable as they promote the selection and dissemination of antibiotic-resistant bacteria in both the target organisms, as well as in the environment [3]. Therefore, the use of alternative control approaches is becoming increasingly important for further development of more sustainable aquaculture practices. In recent years, heat shock proteins (Hsps) have received considerable attention owing to their multi-functional features [4,5]. They are soluble intracellular chaperones present in all organisms from prokaryotes to eukaryotes [6,7]. Most Hsps are constitutively expressed under normal physiological conditions, however, their expression is up-regulated by various physiological stressors, such as high temperature, toxins, osmotic stress, ultra- violet and gamma radiation, certain chemicals and drugs, hypoxia, glucose deprivation, and microbial infection that could potentially damage the cellular and molecular structures in the cells [8,9]. Hsps perform essential biological functions under both normal and stressful conditions such as assisting in the folding of nascent proteins, translocation of these proteins between cell organelles, assembly and disassembly of multi-subunit complexes, refolding or degradation of denatured proteins due to stresses, dissolution of pathological protein aggregates, and other processes enhancing the survival of normal and diseased cells and tissues [10]. Evidence from several studies suggested that Hsps, particularly those of the Hsp70 family, can mediate the generation of strong innate and adaptive immune responses against many diseases [6,7,11], leading to the formulation of strategies to fight infections. Recently, our laboratory has explored the hypothesis that Hsps control disease in aquaculture and found that induction of Hsp in Artemia (eukaryotic Hsp70) through a non-lethal heat shock is associated with protection against virulent Vibrios [12]. In another study [13], the same authors observed that ingestion of Escherichia coli overproducing DnaK (prokaryotic equivalent to Hsp70) signif- icantly improved the survival of gnotobiotically cultured Artemia upon challenge with V. campbellii. These studies suggest that Hsp70 (either prokaryotic or eukaryotic) conferred protection to Vibrio- challenged Artemia. The Hsp70 family members derived either from the prokaryotes or eukaryotes have a high degree of sequence * Corresponding author. Tel.: þ32 926 43754; fax: þ32 926 44193. E-mail address: peter.bossier@ugent.be (P. Bossier). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.07.011 Fish & Shellfish Immunology 29 (2010) 733e739