International Journal of Medical Microbiology 301 (2011) 97–104 Contents lists available at ScienceDirect International Journal of Medical Microbiology journal homepage: www.elsevier.de/ijmm Viral hemagglutinin is involved in promoting the internalisation of Staphylococcus aureus into human pneumocytes during influenza A H1N1 virus infection Claudio Passariello a,∗ , Lucia Nencioni a , Rossella Sgarbanti a , Danilo Ranieri b , Maria Rosaria Torrisi b , Sandro Ripa c , Enrico Garaci d , Anna Teresa Palamara e,f a Dept. of Public Health Sciences, University of Rome “La Sapienza”, Rome, Italy b Institute Pasteur, Cenci Bolognetti Foundation, Department Experimental Medicine, University of Rome “La Sapienza”, Rome, Italy c School of Pharmacy, Microbiology Unit, University of Camerino, Camerino, Italy d Dept. of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy e Institute Pasteur, Cenci Bolognetti Foundation, Department of Public Health Sciences, University of Rome “La Sapienza”, Rome, Italy f IRCSS San Raffaele La Pisana, Rome, Italy article info Article history: Received 20 November 2009 Received in revised form 28 June 2010 Accepted 28 June 2010 Keywords: Influenza Staphylococcus aureus Secondary bacterial pneumonia Hemagglutinin Internalisation abstract Secondary pneumonia caused by Staphylococcus aureus is reemerging as a primary cause of excess mor- tality associated with infection by the influenza A virus. We have investigated in vitro the cellular and molecular mechanisms underlying this synergism. Experimental data show a significant increase in the efficiency of internalisation of S. aureus into cultured pneumocytes during the early phases of viral infec- tion, while a relevant increase in the efficiency of adhesion is evident only later during viral infection, suggesting that the 2 effects are based on different molecular mechanisms. Data reported in this paper show that S. aureus cells can bind the viral hemagglutinin (HA) and that this binding promotes enhanced bacterial internalisation by 2 mechanisms: binding to HA exposed at the surface of infected cells and binding to free extracellular virions, enabling internalisation at high efficiency also in cells that are not infected by the virus. The affinity of binding that involves S. aureus and HA was shown to be enhanced by the reducing extracellular environment that the virus can generate. © 2010 Elsevier GmbH. All rights reserved. Introduction The occurrence of secondary bacterial pneumonia (SBP) in patients infected by the influenza A virus (IAV) became of dramatic actuality following spread of the Spanish pandemic of 1918, when about 8 million patients died for a pneumonia caused by Strepto- coccus pneumoniae or Staphylococcus aureus (S. aureus)(Maxwell et al., 1949). SBP has been a prominent cause of mortality also dur- ing the pandemics of 1957 and 1968 and during common epidemic influenza seasons (Simonsen et al., 1997, 1998, 2000). Although a number of common mechanisms were invoked to explain the occurrence of SBP, much attention was devoted to specific syn- ergisms, as the one involving IAV and Str. pneumoniae, in which the viral neuraminidase promotes bacterial adherence to epithelial cells (McCullers, 2006). Similarly, the ability of Str. pyogenes to bind the viral hemagglutinin (HA) triggers the ability of this bacterium to promote a lethal invasive infection (Okamoto et al., 2003, 2004). ∗ Corresponding author at: Dipartimento di Scienze di Sanità Pubblica, Sezione di Microbiologia Farmaceutica, Università di Roma “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy. Tel.: +39 06 49914885; fax: +39 06 4451324. E-mail address: claudio.passariello@uniroma1.it (C. Passariello). In the last epidemic seasons S. aureus reemerged as a major cause of influenza-associated SBP (CDCP, 2004; Finelli et al., 2008; Podewils et al., 2005). The enhanced prevalence of staphylococcal pneumonia, coupled with the dramatic rise in invasive community- acquired S. aureus infections, mostly sustained by highly virulent, methicillin-resistant strains, and with the diffusion of a new influenza pandemic (Cheng et al., 2009; CDCP, 2009), strongly sup- port the need for more detailed data on this cooperation, in order to develop public health strategies for 2 phenomena that may collide with devastating effects. A characterisation of the cooperation involving IAV and S. aureus was repeatedly attempted in the past (Davison and Sanford, 1981, 1982; El Ahmer et al., 1999; Sanford and Ramsay, 1986; Selinger et al., 1981). All these studies agreed that infection with different influenza viruses promotes adhesion of S. aureus to infected cells. Nevertheless, they could not clearly identify the molecular compo- nents involved in the cooperation, although they demonstrated that unidentified bacterial components interact with proteins shared by both infected and non-infected cells or unique to virus-infected cells (Sanford and Ramsay, 1986). Recently Braun et al. (2007) developed an animal model to study this cooperation and demon- strated that this coinfection promotes bacterial dissemination and results in a synergistic amplification of the inflammatory reac- 1438-4221/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2010.06.008