HadA is an atypical new multifunctional trimeric coiled-coil adhesin of Haemophilus influenzae biogroup aegyptius, which promotes entry into host cells Davide Serruto, 1‡ Tiziana Spadafina, 1‡ Maria Scarselli, 1 Stefania Bambini, 1 Maurizio Comanducci, 1 Sonja Höhle, 2 Mogens Kilian, 2 Esteban Veiga, 3,4,5† Pascale Cossart, 3,4,5 Marco R. Oggioni, 6 Silvana Savino, 1 Ilaria Ferlenghi, 1 Anna Rita Taddei, 7 Rino Rappuoli, 1 Mariagrazia Pizza, 1 Vega Masignani 1 and Beatrice Aricò 1 * 1 Research Center, Novartis Vaccines and Diagnostics, 53100 Siena, Italy. 2 Department of Medical Microbiology and Immunology, Aarhus University, The Bartholin Building, DK-8000 Aarhus, Denmark. 3 Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris F-75015, France. 4 INSERM, U604, Paris F-75015, France. 5 INRA, USC2020, Paris F-75015, France. 6 Laboratorio di Microbiologia Molecolare e Biotecnologia, Dipartimento di Biologia Molecolare, University of Siena, 53100 Siena, Italy. 7 Centro Interdipartimentale di Microscopia Elettronica, University of Tuscia, 01100 Viterbo, Italy. Summary The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotrans- porter adhesins, which includes structurally related proteins, such as YadA of Yersinia entero- colitica and NadA of Neisseria meningitidis. In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA (Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is pre- dicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF. Introduction The recently recognized ‘oligomeric coiled-coil adhesin’ (Oca) family belongs to the wider family of trimeric autotransporter adhesins (TAAs) characterized by the ability to form highly stable trimers on the bacterial surface and by a common mechanism of secretion, which is linked to their trimerization (Surana et al., 2004; Cotter et al., 2005; Linke et al., 2006). To date, all of them appear to have adhesive activity that is involved in mediating bacterial interaction with either host cells or extracellular matrix (ECM) proteins and in some cases in inducing invasion of target cells (Yang and Isberg, 1993; McMichael et al., 1998; Eitel and Dersch, 2002; Laarmann et al., 2002; Ray et al., 2002; Roggenkamp et al., 2003; Li Received 25 September, 2008; revised 19 February, 2009; accepted 20 February, 2009. *For correspondence. E-mail beatrice.arico@ novartis.com; Tel. (+39) 0577 243088; Fax (+39) 0577 243564. † Present address: Dto. Inmunología, primera planta, Hospital de la Princesa, 28006 Madrid, Spain. ‡ These authors contributed equally to this work. Cellular Microbiology (2009) 11(7), 1044–1063 doi:10.1111/j.1462-5822.2009.01306.x First published online 27 March 2009 © 2009 Blackwell Publishing Ltd cellular microbiology