Role of connexin-43 hemichannels in the pathogenesis of Yersinia enterocolitica L.A. Velasquez Almonacid a , S. Tafuri b,c , L. Dipineto a , G. Matteoli a , E. Fiorillo b,c , R. Della Morte b,c , A. Fioretti a , L.F. Menna a , N. Staiano b,c, * a Dipartimento di Patologia e Sanità Animale, Università di Napoli Federico II, via F. Delpino 1, 80137 Napoli, Italy b Dipartimento di Strutture, Funzioni e Tecnologie Biologiche, Università di Napoli Federico II, via F. Delpino 1, 80137 Napoli, Italy c Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, via S. Pansini 5, 80131 Napoli, Italy article info Article history: Accepted 7 August 2008 Keywords: Yersinia enterocolitica Connexin-43 HeLa cell Uptake Signalling abstract Connexin (Cx) channels are sites of cytoplasmic communication between contacting cells. Evidence indi- cates that the opening of hemichannels occurs under both physiological and pathological conditions. In this paper, the involvement of Cx-43 hemichannels is demonstrated in the pathogenesis of Yersinia. Parental HeLa cells and transfected HeLa cells stably expressing Cx-43 (HCx43) were infected with Yer- sinia enterocolitica, and bacterial uptake was measured by the colony-forming unit method. Bacterial uptake was higher in HCx43 cells than in parental cells and was inhibited by the Cx channel blocker, 18-a-glycyrrhetinic acid (AGA). The inhibitory effect of AGA was more pronounced on the Y. enterocolitica uptake by HCx43 cells than by parental cells. The ability of HCx43 cells to incorporate the permeable fluo- rescent tracer Lucifer Yellow (LY) was assessed. Dye incorporation was inhibited by AGA, whereas Y. enterocolitica infection of HCx43 cells increased LY incorporation. Western blotting analysis demon- strated that Y. enterocolitica infection of HCx43 cells induced tyrosine phosphorylation of Cx-43, thus supporting a critical role for Cx-43 in the strategies exploited by bacterial pathogens to invade non- phagocytic cells. Ó 2008 Elsevier Ltd. All rights reserved. Introduction The Gram-negative pathogen Yersinia enterocolitica causes a broad range of gastrointestinal syndromes ranging from acute enteritis and enterocolitis to mesenteric lymphadenitis (Cover and Aber, 1989; Fredriksson-Ahomaa et al., 2006). After ingestion, the bacteria transported to the terminal ileum adhere to the intes- tinal epithelium overlying the lymphoid follicles of Peyer’s patches (Hamzaoui et al., 2004). Subsequently, the bacteria invade the host epithelium and proliferate in the underlying lymphoid tissue. Like many other pathogens, Yersinia bacteria have evolved strat- egies to evade phagocytosis by host immune cells such as macro- phages and neutrophils (Aldridge et al., 2005; Galàn and Cossart, 2005; Bonazzi and Cossart, 2006; Pizarro-Cerda and Cossart, 2006). Resistance to host defence is due to the 70–75 kb virulence plasmid encoding for a type III secretion apparatus along with effec- tor proteins known as Yops (Weidow et al., 2000; Deleuil et al., 2003; Fallman and Gustavsson, 2005; Viboud and Bliska, 2005). Yersinia bacteria can be also engulfed by cells that are generally considered non-phagocytic, including intestinal epithelial cells, hepatocytes and endothelial cells (Aldridge et al., 2005; Galàn and Cossart, 2005; Pizarro-Cerda and Cossart, 2006). Efficient entry into non-phagocytic cells by Y. enterocolitica is mediated by the 108 kDa outer membrane protein, invasin, which binds the b 1 chain of inte- grin receptors on the mammalian cell surface (Isberg and Tran Van Nhieu, 1994; Gustavsson et al., 2002; Scibelli et al., 2005; Wong and Isberg, 2005). Furthermore, another Yersinia invasin, YadA, is also able to interact with b1 integrins, thus promoting an efficient up- take pathway (Boyle and Finlay, 2003). Bacterial engagement of integrins on host cell surface triggers signals resulting in bacterial internalization (Alrutz and Isberg, 1998; Weidow et al., 2000; Bruce-Staskal et al., 2002; Hudson et al., 2005) so allowing bacteria to establish persistent infection and promote invasion. More recently, a role for the endocytosis machinery in Yersinia entry in non-phagocytic cells has been demonstrated, suggesting that a clathrin-dependent endocytic mechanism is also involved in the internalization of these bacteria into host cells (Tran Van Nhieu et al., 1996; Veiga and Cossart, 2006). Other studies have pro- vided evidence that bacterial invasion and dissemination involve the opening of connexin (Cx) channels. In particular, Shigella inva- sion has been shown to induce the opening of Cx-26 hemichannels in an actin- and phospholipase C-dependent manner (Tran Van Nhieu et al., 2003). Furthermore, Staphylococcus aureus-derived peptidoglycan induces Cx-43 expression and functional gap junc- tion intercellular communication in microglia (Garg et al., 2005). Hemichannels, also known as connexons, are made up by a hex- americ array of proteins encoded by the Cx multi-gene family 1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.08.011 * Corresponding author. Address: Dipartimento di Strutture, Funzioni e Tecnol- ogie Biologiche, Università di Napoli Federico II, via F. Delpino 1, 80137 Napoli, Italy. Tel.: +39 0812536108; fax: +39 0812536097. E-mail address: staianor@unina.it (N. Staiano). The Veterinary Journal 182 (2009) 452–457 Contents lists available at ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl