Cellular machinery to fuse antimicrobial autophagosome with lysosome

Abstract

Autophagy is an intracellular bulk degradation/recycling system that turns over cellular constituents and also functions to degrade intracellular foreign microbial invaders by a process termed xenophagy (antimicrobial autophagy). We previously showed that intracellular group A Streptococcus (GAS) organisms are captured by xenophagosomes, then degraded following fusion with lysosomes. Very recently, we analyzed the molecular mechanism underlying xenophagosome/lysosome fusion and found that endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) were involved. Knockdown of the combinational SNARE proteins Vti1b and VAMP8 with siRNAs disturbed autophagic fusion with lysosomes, and cellular bactericidal efficiency was significantly diminished. Furthermore, knockdown of those SNAREs inhibited the fusion of canonical autophagosomes with lysosomes. In addition, important findings showed that Vti1b is derived from autophagic compartments, whereas VAMP8 originates from lysosomes. Together, these results strongly suggest that SNARE proteins Vti1b and VAMP8 mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.

Keywords: SNARE; VAMP8; Vti1b; autophagy; xenophagy.

Figure 1

Knockdown of VAMP8 and Vti1b inhibits colocalization of xenophagosomes with lysosomes. HeLa cells expressing GFP-LC3 were transfected with control siRN A, and VAMP8 and Vti1b siRN As. Following infection with GAS, the cells were fixed and incubated with anti-LAMP1 antibodies and observed with a confocal microscope. Cellular and bacterial DN A were stained with DAPI. LAMP1 failed to colocalize with GFP-LC3 in the SNARE -depleted cells. Boxed regions in the upper panels show magnifications of the lower panels. Bars indicate 5 µm.

Figure 2

SNARE proteins VAMP8 and Vti1b fuse xenophagosomes/canonical autophagosomes with lysosomes. Intracellular GAS organisms are entrapped within xenophagosomes, then xenophagosomes undergo a stepwise maturation process that consists of a fusion event with lysosomes, which allows the autophagic vacuole to acquire lysosomal proteases. That fusion is mediated by a combinational complex of VAMP8 and Vti1b from xenophagosomes. Finally, GAS organisms are degraded by xenophagosomes (autolysosomes) possessing lysosomal degradation enzymes. The complex of VAMP8 and Vti1b also mediates the fusion between canonical autophagosomes and lysosomes. Vti1b originates from autophagic vacuoles, whereas VAMP8 is recruited from lysosomes to autophagic compartments prior to fusion.