ErbB2 supports N. meningitidis invasion | Hoffmann et al. 139 Figure 7. ErbB2 activation is not required for cortactin recruit- ment within cortical plaques. Cells were pretreated for 2 h with either 5 M AG1478 or 30 M Y27632. Cells noninfected or infected for 3 h with the 2C43 wild-type strain of N. meningitidis were double-stained for ErbB2 (left) and cortactin (middle). Merged images (overlay) of the same fields are presented on the right. fect the tyrosyl phosphorylation induced by N. meningitidis (Fig. 1), had no effect on bacterial entry. Thus, these experi- ments indicate that the ErbB2/src pathway is involved in N. meningitidis internalization into endothelial cells. Since we recently showed that the small Rho GTPases were involved in cortical plaque formation and subsequent N. meningitidis internalization into endothelial cells (unpub- lished data), we analyzed the respective contributions of both ErbB2/src and Rho GTPase–dependent signaling path- ways on bacterial entry into endothelial cells. As shown in Fig. 8 B, treatment with PP2 in the presence of Y27632 al- most totally prevented bacterial entry (90% inhibition), whereas Y27632 alone inhibited bacterial entry by 60%. None of these treatments affected bacterial adhesion to en- dothelial cells (unpublished data). Together, these data dem- onstrate that N. meningitidis promotes two distinct signaling pathways, the ErbB2/src and Rho-GTPase pathways, which cooperate to promote N. meningitidis internalization into endothelial cells. Discussion We have investigated here the tyrosine phosphorylation events occurring during the interaction of N. meningitidis with human endothelial cells. We demonstrate that the acti- vation of the ErbB2 receptor tyrosine kinase is directly in- Figure 8. (A) Entry levels of the different strains of N. meningitidis in HBMECs. HBMECs were infected with the wild-type (WT), the PilT-defective mutant (PilT ), or the PilE-defective mutant (PilE ) of the 2C43 strain of N. meningitidis. After 4 h, the number of internal- ized bacteria in relation to the number of adherent bacteria was determined. (B) Effects of PP2, AG1478, AG1296, and Y27632 on N. meningitidis entry in HBMECs. Cells were either left untreated (NT) or pretreated for 2 h with 5 M PP2, 5 M AG1478, 5 M AG1296, 30 M Y27632, or with both inhibitors PP2 AG1478 or PP2 Y27632, as indicated. Cells were infected with the wild-type strain of N. meningitidis for 4 h in the presence of the inhibitors. The percentage of internalized bacteria in relation to the entry or adhesion of the wild-type strain in absence of any activator or inhibitor was determined, and average values ( SEM) from four independent experiments are presented. volved in host signal transduction promoted by N. meningi- tidis interaction and subsequent bacterial internalization. N. meningitidis induces a massive recruitment and tyro- sine phosphorylation of ErbB2. Our data suggest that the in- teraction of N. meningitidis with human endothelial cells leads to the activation of ErbB2, most likely via formation of ErbB2 homodimers, since no recruitment nor tyrosine phos- phorylation of any other known ErbB family members was detected. We believe that these results constitute an impor- tant finding since, although no soluble ligand for ErbB2 has been identified so far, this receptor appears to be the pre- ferred heterodimerization partner of all ErbB proteins (Tza- har et al., 1996; Graus-Porta et al., 1997). Although the for- mation of most heterodimers is mediated by ErbB ligands, ErbB2-containing heterodimers may also be triggered by other classes of receptors through ligand-independent path- ways (Qiu et al., 1998). ErbB2 homodimerization was previ-