Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAP kinases Laura Canesi a, * , Michele Betti b , Caterina Ciacci b , Alfonso Scarpato b , Barbara Citterio b , Carla Pruzzo c , Gabriella Gallo d a Istituto di Scienze Fisiologiche, Universita Á di Urbino, Loc. Crocicchia, Urbino, 61029 PU), Italy b Ist Scienze Tossicologiche, Igienistiche ed Ambientali, Universita Á di Urbino, Urbino, Italy c Istituto di Microbiologia, Universita Á di Ancona, Italy d Dipartimento Biologia Sperimentale Ambientale ed Applicata, Universita Á di Genova, Italy Received 1 May 2001; received in revised form 3 September 2001; accepted 5 September 2001 Abstract In this work the mechanisms of transduction triggered in Mytilus galloprovincialis hemocytes by bacterial challenge were investigatedinaninvitromodelofinfectionofhemocytemonolayerswith Escherichia coli.Westernblotanalysesofhemocyte extracts with phospho-speci®c anti-MAPK Mitogen Activated Protein Kinase) antibodies indicate that E. coli induced a time dependentactivationofdifferentclassesofMAPKs,mainlyofthestress-activatedp38MAPK.P38activationwascon®rmedby the use of the selective kinase inhibitor SB203580. Moreover, hemocyte pretreatment with SB203580 signi®cantly reduced bacterial killing, whereas PD98059, an inhibitor of extracellularly regulated kinase ERK) activation, was ineffective. Inter- estingly, the PI3-kinase phosphatidylinositol-3-OH-kinase) inhibitor, Wortmannin, reduced both p38 activation and bacterial killing, indicating a critical role also for this lipid kinase in the hemocyte immune response. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bivalve immunity; Cell signaling; Mytilus hemocytes; Escherichia coli; Mitogen activated protein kinases; p38; Bacterial killing 1. Introduction In invertebrate organisms, immunity involves both cell-mediated and humoral systems that operate in a coordinated way to provide protection from invading pathogens [1]. In bivalve molluscs, such as Mytilus sp., cellular responses are carried out by circulating hemocytes, which represent the ®rst line of defence against bacterial infection [2]. Hemocytes can kill microbes through phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes [3,4]andtherespiratoryburstwhichinvolvesproduc- tion of oxygen metabolites [5,6]. Even though these responses have been widely described in bivalves as wellasinmanyinvertebratesystems,thebiochemical events involved in the transduction of bacterial invasion signals into physiological defense responses in invertebrate hemocytes are still unknown. In mammalian systems, activation of a complex Developmental and Comparative Immunology 26 2002) 325±334 0145-305X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII:S0145-305X01)00078-7 www.elsevier.com/locate/devcompimm * Corresponding author. Tel.: 139-0722-304-267; fax: 139- 0722-304-226. E-mail address: canesi@uniurb.it L. Canesi).