Anchoring of perforin-2 via the transmembrane domain is required for antigen escape into the cytosol

Abstract

Perforin-2 is a pore-forming protein localised to the endocytic compartments of dendritic cells and macrophages. It is reported to perform two distinct functions during immune responses: attacking intravacuolar pathogens, and forming pores in endocytic compartments to enable cytosolic delivery of antigens during cross-presentation. The molecular mechanisms that regulate perforin-2 remain unknown. Here, we address how cross-presenting dendritic cells control pore formation in phagosomes while maintaining the integrity of their endocytic compartments. We demonstrate that perforin-2 undergoes extensive proteolytic processing involving multiple endocytic proteases. Although the transmembrane anchor has been proposed to protect host membranes by orienting pores towards bacterial targets, we find that endocytic escape is mediated by full-length, membrane-anchored perforin-2 rather than by the proteolytically released ectodomain. Moreover, we show that perforin-2-mediated antigen translocation does not require low pH, explaining how perforin-2 can form pores in cross-presenting dendritic cells which do not acidify their phagosomes. Our findings point to a critical role of the transmembrane anchor in perforin-2 biology and suggest that perforin-2 employs distinct mechanisms of pore formation during anti-bacterial defence and cross-presentation. Perforin-2 forms pores with functions in anti-bacterial immunity and in antigen delivery into the cytosol during cross-presentation. This study shows that perforin-2 undergoes extensive proteolytic processing, but its full-length form mediates antigen trafficking in dendritic cells. Perforin-2 undergoes extensive proteolytic processing, including ectodomain shedding. Proteolytic maturation of perforin-2 is mediated by a range of proteases, including cathepsins, AEP, and γ-secretase. Proteolytic processing of perforin-2 takes place primarily in low-pH compartments. Endocytic escape of antigens in dendritic cells is mediated by full-length, transmembrane-anchored perforin-2, and does not require low pH. Perforin-2 undergoes extensive proteolytic processing, including ectodomain shedding. Proteolytic maturation of perforin-2 is mediated by a range of proteases, including cathepsins, AEP, and γ-secretase. Proteolytic processing of perforin-2 takes place primarily in low-pH compartments. Endocytic escape of antigens in dendritic cells is mediated by full-length, transmembrane-anchored perforin-2, and does not require low pH. Perforin-2 employs distinct mechanisms of pore formation during anti-bacterial defence and antigen cross-presentation.