Vamp3/syntaxin 4 mediates the basolateral membrane fusion of TfR transcytosis across the BBB and is exploited by pathogenic .

Transcytosis across the blood-brain barrier (BBB), composed of brain microvascular endothelial cells (HBMECs), is tightly controlled to prevent the entry of macromolecules, microorganisms, and toxins into the brain. Transferrin receptor (TfR) transcytosis, one of the few active transcytosis pathways in HBMECs, is extensively utilized for drug transport across the BBB and employed by meningitis-causing bacteria to penetrate into the brain. However, the molecular mechanism facilitating the fusion of TfR vesicles with the basolateral membrane of HBMECs, the final step of transcytosis, has not been experimentally studied. Here, we found that the interaction between the v-SNARE protein VAMP3 on TfR vesicles and the t-SNARE protein syntaxin 4 that is limited to the basolateral membrane in HBMECs mediates the fusion. We also provided evidence that this step is critical for the efficiency of TfR transcytosis. Furthermore, we showed that neonatal meningitis (NMEC) infection significantly boosts the efficiency of this transcytosis by enhancing the expression of VAMP3 and syntaxin 4 through the TLR4-TRAM-TRIF-TRAF3-IKK-IRF3 signaling pathway in HBMECs. Silence or overexpression of VAMP3 and syntaxin 4 reduces or enhances the transcytosis of transferrin and NMEC in a human BBB model . Consistently, the penetration of transferrin and NMEC into the brain was significantly inhibited in VAMP3-deficient mice. These findings provide insights for improving strategies to deliver drugs into the brain and developing effective therapy for meningitis caused by NMEC.