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 <i>Streptococcus</i> (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 <i>RABGEF1</i> 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.

巨自噬（Macroautophagy/autophagy）可通过直接降解入侵病原体的方式在免疫应答中发挥关键作用，这类入侵病原体包括A群链球菌（Group A Streptococcus，GAS），该过程被命名为异体吞噬（xenophagy）。我们此前的研究证实，靶向GAS的自噬泡——被称为含GAS自噬样泡（GcAVs）——以循环内体（recycling endosomes，REs）作为膜来源。然而，GcAVs与REs之间融合的精确分子机制仍未阐明。本研究证实，突触融合蛋白6（syntaxin 6，STX6）被招募至GcAVs，并与VTI1B及VAMP3形成复合物，以调控异体吞噬所必需的GcAV-RE融合过程。STX6通过其基于酪氨酸的分选基序与跨膜结构域靶向GcAV膜，并通过其H2 SNARE结构域定位于GcAVs上转铁蛋白受体（transferrin receptor，TFRC）阳性的点状结构。敲低与敲除实验表明，STX6对于GcAVs与REs的融合以促进自噬清除胞内GAS是必需的。此外，VAMP3与VTI1B可与STX6相互作用，并定位于GcAVs上的TFRC阳性点状结构，同时也参与RE-GcAV融合过程。进一步研究发现，敲除RABGEF1会损害RE-GcAV融合以及STX6-VAMP3的相互作用。上述研究结果表明，RABGEF1通过STX6-VAMP3-VTI1B复合物介导RE与GcAVs的融合，并揭示了针对细菌感染的自噬体形成过程中所涉及的SNARE蛋白动态调控机制。