TRPA1 CHANNELS: MOLECULAR SENTINELS OF CELLULAR STRESS AND TISSUE DAMAGE F elix Viana. Instituto de Neurociencias de Alicante, UMH-CSIC, Avenida S. Ram on y Cajal s.n., 03550, San Juan de Alicante, Spain E-mail address: felix.viana@umh.es TRPA1 is a calcium permeable, non-selective cation channel expressed in mammalian peripheral pain receptors, with a major role in chemo- nociception. TRPA1 has also been implicated in noxious cold and me- chanical pain sensation. TRPA1 has an ancient origin and plays important functions in lower organisms, including thermotaxis, mechano- transduction and modulation of lifespan. Recent work from many labo- ratories has defined the critical role of TRPA1 as a multipurpose sensor of physical (e.g. u.v. light) and chemical (e.g. lipid peroxidation products) harmful signals in multiple barriers tissues, including the gut, the lung and the skin. Here we highlight the identification of TRPA1 as a sensor for lipopolysaccharide (LPS), a toxic byproduct of Gram-negative bacterial lysis. Application of exogenous LPS exerted a rapid (seconds) activation of mouse somatic and visceral nociceptors. These actions were mediated by opening of TRPA1 channels leading to neuronal depolarization, firing of action potentials and rapid elevation of intracellular calcium levels. Human TRPA1 channels, expressed in HEK293 cells, showed a similar sensitivity to LPS. The intradermal injection of LPS produced a rapid (minutes) inflam- matory response that was accompanied by pain and acute vascular re- actions, including neurogenic inflammation and CGRP release (measured by enzyme immunoassay). These responses were severely blunted in TRPA1 KO mice and developed independently of TLR4 activation. More- over, the capacity of various forms of LPS, purified from different patho- gens, to activate TRPA1 in vitro correlated with their ability to induce inflammatory responses in vivo. In summary, these findings point to TRPA1 channels as key molecular determinants of rapid LPS effects on sensory neurons and their terminals, leading to acute neurogenic inflammation and pain. These results suggest that TRPA1 channels may play a previously unrecognized role in the first line of immune defense against microbial pathogens. Pharmacological targeting of TPA1 channels could represent a novel therapeutic avenue for treatment of complications derived from Gram negative bacterial infections, including septic shock. Keywords: LPS, Bacterial endotoxin, Pain, Inflammation HOST IMMUNOMODULATION BY MYCOLACTONE, THE MYCOBACTERIUM ULCERANS TOXIN Jean-David Morel, Ludivine Baron, Laure Guenin-Mac e, Caroline Demangel * . United’Immunobiologie de l’Infection, Institut Pasteur, Inserm U1221, Paris, France * Corresponding author. E-mail address: caroline.demangel@pasteur.fr (C. Demangel). Why infected individuals fail to mount protective immune responses against Mycobacterium ulcerans is the subject of an intense research that aims to better control the emergence of Buruli ulcer disease, and discover new means of manipulating immunity. M. ulcerans virulence relies on bacterial production of mycolactone, a polyketide toxin with tissue-specific proper- ties. In addition to inducing local skin ulceration and analgesia, mycolactone diffuses in infected hosts to dampen cellular immune responses at the sys- temic level. This seminar will present recent work identifying the Sec61 translocon as the host receptor targeted by mycolactone, and illustrating the potential of mycolactone for tuning inflammatory and immune responses 1 . 1 Baron et al. (2016) Mycolactone subverts immunity by selectively blocking the Sec61 translocon. J. Exp. Med. in press. Keywords: Mycolactone, Mycobacterium ulcerans, Buruli ulcer, Immune response, Sec61 translocon. ACTIN ACTIVATES PSEUDOMONAS AERUGINOSA EXOY NUCLEOTIDYL CYCLASE TOXIN Alexander Belyy * , Daniel Ladant, Undine Mechold. Institut Pasteur, CNRS URA3528, Unite de Biochimie des Interactions macromoleculaires, Departement de Biologie Structurale et Chimie, Paris, France * Corresponding author. E-mail address: alexander.belyy@pasteur.fr (A. Belyy). ExoY is a nucleotidyl cyclase toxin from the opportunistic human pathogen Pseudomonas aeruginosa. Once injected into the target cell by the Type 3 secretion system (T3SS), the toxin needs to be activated by a eukaryotic cofactor, which was recently identified as F-actin. Actin from budding yeast Saccharomyces cerevisiae is encoded by the single ACT1 gene; the protein is 90% identical to mammalian beta-actin, and can activate ExoY in vitro and in vivo, the latter results in yeast lethality. To find key residues in the ExoY- actin interaction, we prepared a library of yeast cells with randomly mutagenized actin molecules. The library was transformed with an ExoY- coding plasmid and the toxin was expressed. Actin sequencing of surviving cells revealed mutations in Asp25, which is localized on the surface of F- actin. Lysates prepared from these strains did not activate the toxin in vitro. Loss of the activation after a single amino acid substitution strengthens the point, that actin is the only activator of the ExoY toxin from P. aeruginosa. Keywords: ExoY, Actin, T3SS CHARACTERIZATION OF AM IT, AN ANTI-INSECT b-TOXIN ISOLATED FROM THE VENOM OF SCORPION ANDROCTONUS MAURETANICUS Khadija Daoudi a, b , Myriam Rezzak a , Oussama Bourouah a , Fatima Chgoury a , Naoual Oukkache a, * . a Laboratory Venoms & Toxins, Pasteur Institute of Morocco, 1 Place Louis Pasteur, Casablanca, 20360, Morocco; b Laboratory of Physiology and Molecular Genetics, Department of Biology, Faculty of Science Ain Chock, B.P 5366, Maarif, Casablanca, Morocco * Corresponding author. E-mail address: oukkache.naoual@gmail.com (N. Oukkache). In the present study, a novel toxin, called Am IT from the venom of scorpion Androctonus mauretanicus is isolated and characterized. A detailed analysis of the action of Am ITon insect axonal sodium currents is reported. Am IT was purified through gel filtration followed by C18 reversed-phase HPLC. Toxicity of Am IT in vivo was assessed on male German cockroach (Blattella ger- manica) larvae and C57/BL6 mice. Cross-reactivity of Am IT with two b-toxins was evidenced using 125 I-iodinated toxin-based radioimmunoassays with synaptosomal preparations from rat brain. The complete amino acid sequence of Am IT was finally determined by Edman sequencing. Am IT was observed to compete with AaH IT4 purified from the venom of scorpion Androctonus australis in binding assays. It was recognized by an antibody raised against a b-type toxin, which indicated some structural similarity with b-toxins (or related toxin family). The novel toxin exhibited dual activity since it competed with anti-mammal toxins in binding assays as well as showed contracting activity to insect. The toxin competed with radio-labeled b-toxin Css IV by binding to Na + channels of rat brain synaptosomes. Analysis of toxin amino acid sequences showed that Am IT shares high structural identity (92%) with AaH IT4. In conclusion, Am IT not only reveals an anti- insect compound properties secreted by 'Old World' scorpions, paralyzing insect larvae by binding to Na + channels on larvae's nerve-cell membranes, but also exerts toxic activity in mice, which is similar to anti-mammal toxins from 'New World' scorpions (North and South Americas). Therefore, Am IT appears to be structurally and functionally similar to AaH IT4. Keywords: Scorpion venom, Beta-insect, Toxin CRYSTAL STRUCTURE AND FUNCTIONAL DOMAINS OF THE MAMBAQUARETIN-1, A VASOPRESSIN TYPE 2 RECEPTOR PEPTIDE INHIBITOR FOR KIDNEY CYSTS TREATMENT Laura Droctov e a, * , Justyna Ciolek a , Manon Lancien a , Enrico A. Stura a , Laura Vera a , Nicolas Floquet b , Ralph Witzgall c , Bernard Mouillac d , Christiane Mendre d , Gilles Mourier a , Denis Servent a , Nicolas Gilles a . a Service d’ingenierie moleculaire des proteines (SIMOPRO), IBITECS, CEA Universite Paris-Saclay, F-91191, Gif-sur-Yvette, France; b Institut des Biomolecules Max Mousseron, Faculte de Pharmacie, 15 avenue Charles Flahault, 34093, Montpellier cedex 5, France; c Institute for molecular and cellular anatomy, University of Regensburg, Universit€ atsstr. 31, 93053, Regensburg, Germany; d Institut de Genomique Fonctionnelle, UMR5203 CNRS U1191 INSERM, UM1 UM2, 141 rue de la cardonille, 34094, Montpellier, France Abstracts / Toxicon 149 (2018) 86e97 91