ErbB2 supports N. meningitidis invasion | Hoffmann et al. 141 Figure 9. Schematic representation of the signaling pathways activated by N. meningitidis and involved in bacterial en- try into endothelial cells. (1) Type IV pili initiate the interaction of virulent, capsu- lated N. meningitidis with human endo- thelial cells by interacting with a cellular receptor that remains to be clearly identi- fied. Bacteria then proliferate, locally forming a colony at their site of attach- ment on the cell surface. (2) This pili- dependent adhesion induces, through a Rho GTPase-independent pathway, the recruitment of ezrin and moesin, two proteins that link the cellular membrane to the actin cytoskeleton, and the cluster- ing of several transmembrane proteins: ErbB2 and the ezrin binding proteins CD44 and ICAM-1. (3) The activation of both Rho and Cdc42 GTPases induces a localized polymerization of cortical actin, leading to the formation of mem- brane projections reminiscent of epithelial microvilli structures, which surround single bacteria and provoke their internal- ization within endothelial intracellular vacuoles. Downstream of bacteria- induced ErbB2 activation are the ras/ MAPK signaling pathway, and the src ty- rosine kinase which phosphorylates cort- actin, and is involved in bacterial entry. cortactin was highly recruited underneath the bacterial colo- nies, and that this recruitment was not correlated with ty- rosyl phosphorylation of cortactin, but was instead de- pendent on cortical actin polymerization induced by N. meningitidis. Interestingly, ErbB2 activation induced by EGF, while activating src (unpublished data), did not lead to cortactin phosphorylation. This apparent discrepancy may result from the activation of specific signaling pathways acti- vated by ErbB2 homodimers (in response to N. meningitidis) or ErbB2/EGFR heterodimers (in response to EGF), includ- ing the activation of alternative kinases involved in cortactin phosphorylation, such as Fer (Kim and Wong, 1998). Alter- natively, cortactin phosphorylation induced in infected cells may result from the local concentration of signaling mole- cules in microdomains underneath the bacterial colonies. Further studies are required to investigate the consequences of the tyrosyl phosphorylation of cortactin and to character- ize the mechanism by which the src signaling pathway facili- tates N. meningitidis internalization into endothelial cells. In summary, these results, summarized in Fig. 9, provide evidence for a novel mode of activation and an unsuspected involvement of ErbB2 in host pathogen interaction. We be- lieve that they will help unravel the pathophysiological mechanism of meningitis associated with N. meningitidis in- fection. Materials and methods Antibodies and reagents Antibodies to cortactin (p80/85) and pp60c-src were purchased from Up- state Biotechnology; antiphosphotyrosine antibody (4G10) was from Med- itech; polyclonal antibodies directed against EGFR, ErbB2, ErbB3, ErbB4 were purchased from Santa Cruz Biotechnology, Inc. Antiphospho Erk1/2 (Thr202/Tyr204) was from Cell Signaling Technology. Rhodamine phalloi- din and recombinant human EGF were from Sigma-Aldrich. GST fusion protein containing src-SH2 domain was provided by Dr. S. Fisher (Institut Cochin de Génétique Moléculaire, Paris, France). The selective inhibitors AG1478, AG1296, and PP2 were from Calbiochem, and Y27632 was pro- vided by Yoshitomi Pharmaceutical. Bacterial strains and growth conditions 2C43 (formerly clone 12), a piliated capsulated Opa variant of the sero- group C meningococcal strain 8013, the nonpiliated derivative strain pilE:: Km, and the PilT derivative strain pilT::Erm were described previously (Nassif et al., 1993; Pujol et al., 1999). The strain designated ROU (group W135, ET37), is a piliated, capsulated isolate obtained from the cere- brospinal fluid of a 2-mo-old infant and has been described previously (Pron et al., 1997). Before experiments, N. meningitidis was grown over- night on GCB solid medium (4% GC medium base; Difco), 1% agar, 0.4% glucose, 0.2 mg/ml thiamine, 0.0005% Fe(NO3)39H2O, 0.01% L-glutamine at 37 C under 5% CO2. Several colonies were then selected and grown in DME supplemented with 0.1% BSA for 2 h and were finally diluted to 107 bacteria/ml suspension for cell infection. Cell culture, infection, and quantification of N. meningitidis adhesion or entry into endothelial cells HBMECs (Schweitzer et al., 1997), provided by Dr. B. Weksler (Weill Medical College of Cornell University, Ithaca, NY), were cultured in DME Glutamax (Life Technologies) supplemented with 10% heat-inactivated fe- tal bovine serum, 7.5 g/ml endothelial cell growth supplement (Sigma- Aldrich), 7 UI heparin (Sigma-Aldrich) and 10 mM Hepes. Infections were performed as follows: confluent monolayers of HBMECs were starved for 18 h before infection in DME supplemented with 0.1% BSA (starvation medium) to lower the basal level of tyrosine phosphoryla- tions. HBMECs were then overlaid for 30 min with 107 bacteria in starva- tion medium. Cells were then washed three times with starvation medium to remove nonadherent bacteria and infection was allowed to proceed for various periods of time. To avoid reinfection of the monolayers, cells were washed once every hour with starvation medium. When indicated, cells were pretreated for 2 h with AG1478, AG1296, PP2, or Y27632 before in- fection. Cells were then infected as above and the infection was allowed to proceed for 4 h in the presence of the inhibitors. For adhesion and inter- nalization assays, cells grown to confluency in 6-well plates were infected in starvation medium, washed three times, and the infection was allowed