The STX6-VTI1B-VAMP3 complex facilitates xenophagy by regulating the fusion between recycling endosomes and autophagosomes.

Macroautophagy/autophagy plays a critical role in immunity by directly degrading invading pathogens such as Group A Streptococcus (GAS), through a process that has been named xenophagy. We previously demonstrated that autophagic vacuoles directed against GAS, termed GAS-containing autophagosome-like vacuoles (GcAVs), use recycling endosomes (REs) as a membrane source. However, the precise molecular mechanism that facilitates the fusion between GcAVs and REs remains unclear. Here, we demonstrate that STX6 (syntaxin 6) is recruited to GcAVs and forms a complex with VTI1B and VAMP3 to regulate the GcAV-RE fusion that is required for xenophagy. STX6 targets the GcAV membrane through its tyrosine-based sorting motif and transmembrane domain, and localizes to TFRC (transferrin receptor)-positive punctate structures on GcAVs through its H2 SNARE domain. Knockdown and knockout experiments revealed that STX6 is required for the fusion between GcAVs and REs to promote clearance of intracellular GAS by autophagy. Moreover, VAMP3 and VTI1B interact with STX6 and localize on the TFRC-positive puncta on GcAVs, and are also involved in the RE-GcAV fusion. Furthermore, knockout of RABGEF1 impairs the RE-GcAV fusion and STX6-VAMP3 interaction. These findings demonstrate that RABGEF1 mediates RE fusion with GcAVs through the STX6-VAMP3-VTI1B complex, and reveal the SNARE dynamics involved in autophagosome formation in response to bacterial infection.