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

## TL;DR

Scientists discovered how a protein pathway helps transport molecules and bacteria across the brain barrier, offering new ways to treat brain infections or deliver drugs.

## Contribution

Identified VAMP3 and syntaxin 4 as key proteins mediating TfR transcytosis and exploited by meningitis-causing bacteria.

## Key findings

- VAMP3 and syntaxin 4 mediate the final fusion step of TfR transcytosis across the BBB.
- NMEC enhances transcytosis efficiency by upregulating VAMP3 and syntaxin 4 via the LPS-TLR4 pathway.
- Modulating VAMP3 and syntaxin 4 levels affects drug and bacterial transcytosis in human BBB models.

## Abstract

Transferrin receptor (TfR) transcytosis is an essential strategy for delivering therapeutics into the brain and is exploited by meningitis-causing bacteria to penetrate the blood–brain barrier (BBB). We found that the interaction between VAMP3 on TfR vesicles and syntaxin 4 at the basolateral membrane of brain microvascular endothelial cells mediates the final fusion step of TfR transcytosis. Enhancing the expression of VAMP3 and syntaxin 4 promotes TfR transcytosis across the BBB. Furthermore, neonatal meningitis Escherichia coli (NMEC) increases its transcytosis efficiency by upregulating host VAMP3 and syntaxin 4 expression through the LPS-TLR4 signaling pathway. Our finding suggests that modulating the expression of VAMP3 and syntaxin 4 could be an effective strategy to improve brain drug delivery or to treat NMEC infection.

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 Escherichia coli (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 in vitro. 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.