Vaccine 23 (2005) 2551–2556 The Rho GTPase activators CNF1 and DNT bacterial toxins have mucosal adjuvant properties Patrick Munro a , Gilles Flatau a , Fabienne Anju` ere b , V´ eronique Hofman b,c , Cecil Czerkinsky b , Emmanuel Lemichez a,∗ a INSERM, U627, Facult´ e de M´ edecine, 28 Avenue de Valombrose, F-06107 NiceCedex 2, France b INSERM, EMI 02-15, Facult´ e de M´ edecine, 28 Avenue de Valombrose, F-06107 NiceCedex 2, France c Service d’Anatomo-Pathologie, Centre Hospitalier de Nice, Hopital Pasteur, BP 69, 06002 Nice Cedex 1, France Received 24 June 2004; received in revised form 12 October 2004; accepted 11 November 2004 Available online 8 December 2004 Abstract Cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli belongs to a family of factors activating Rho GTPases. We report the in vivo effects of CNF1 in mice co-fed toxin and the soluble protein antigen ovalbumin (OVA). Similar to cholera toxin, CNF1 elicits adjuvanticity anti-OVA responses, both systemic and mucosal. In contrast, the catalytic inactive mutant CNF1-C866S demonstrated no effects. Using dermonecrotic toxin (DNT), a closely related Rho activating toxin from Bordetella, we discovered that the adjuvant property is within the DNT catalytic domain. Manipulation of Rho proteins thus provides a possible new approach for the development of effective mucosal immunoadjuvants. © 2004 Elsevier Ltd. All rights reserved. Keywords: CNF1; DNT; Rho; Rac; Cdc42; GTPases; Adjuvant; Mucosa 1. Introduction Despite considerable progress in the field of vaccination, the need for more efficient vaccines together with mucosal adjuvants remains a real challenge [1,2]. Bacterial toxins, such as cholera toxin (CT), and related enterotoxins have long been known to be powerful mucosal adjuvants and might serve as mucosal vaccines [3–6]. The cytotoxic necrotizing factor 1 (CNF1) belongs to a family of bacterial toxins including CNF2 from Escherichia coli [7], CNFy from Yersinia pseudotuberculosis [8] and the dermonecrotic toxin (DNT) from Bordetella [9]. CNF1 is a classical A-B protein toxin of 1014 amino acids produced by uropathogenic and meningitis causing E. coli [10]. Both CNF1 and DNT are composed of three different domains, each of them being responsible for one of the three steps of ∗ Corresponding author. Tel.: +33 4 93 37 77 09; fax: +33 4 93 53 35 09. E-mail address: lemichez@unice.fr (E. Lemichez). the intoxication process [11–13]. First, the amino-terminal domain of CNF1 binds to a cell surface receptor and is inter- nalized into endocytic vesicles [11,14,15]. During vesicular trafficking, the translocation domain of CNF1, once reaching acidic conditions, allows injection of the carboxy-terminal catalytic domain through the vesicular membrane into the cytosol [14,16]. Once inside the cytosol, CNF1 deamidates Rho glutamine 63 and its equivalent 61 for Rac and Cdc42 [17–19]. Deamidation or transglutamination of Rho proteins inhibits their GTPase activities thus conferring them dom- inant positive properties [17,18,20]. CNF1 and DNT cat- alytic domains share a high level of amino acids sequence homologies [11,13]. Consistently, it has been demonstrated that DNT toxin deamidates and transglutaminates Rho pro- teins [20,21]. Once activated by CNF1, Rho GTPases are then rapidly processed by ubiquitin-mediated proteasomal degradation [22,23]. This dual counter intuitive mechanism of protein activation and degradation is responsible for mod- erating the activation of Rho proteins [22]. The CNF1 activity 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.11.042