Direct visualization of membrane-spanning pores formed by a Leishmania amazonensis pore-forming cytolysin, as probed by atomic force microscopy

We have previously shown that Leishmania amazonensis produces and secretes a cytolysin that lyses membranes of mammalian cells, including macrophages, its host cell. Using the patch-clamp technique, we have previously demonstrated that the mechanism by which this cytolysin rupture macrophages plasma membrane is by pore formation, which lead us to name it leishporin. While we have characterized leishporin in several aspects, its molecular identity is still unknown. Its behavior suggests that leishporin is, or depend on, a protein, but recent results also suggests that a non-protein molecule is involved in cell lysis. Although the patch-clamp has undeniably revealed that L. amazonensis extracts generates pores in macrophages, these structures have not been spotted on cell membranes, which prompted us to several questions: 1) What is the appearance of leishporin-induced pores? Is it similar to that of other described pores? 2) Do these pores physically span lipid bilayers? 4) Are their directly-measured sizes compatible with those previously suggested by patch-clamp? 5) Do these pores fuse with one another, enlarging in size, as suggested by our previous reports? In the present work, we have used two membrane models, erythrocytes and liposomes, to visualize pores induced by the cytolysin on parasite extracts. Leishporin-mediated lysed erythrocytes or liposomes were analyzed by atomic force microscopy (AFM), which allowed us to visualize multiple membrane-spanning pores of variable diameters, ranging from 25 to 230 nm. They do not resemble to protein-formed pores, but rather, to pores made by small molecules such as lipids or peptides, as also visualized by AFM. Our results suggest that the maximum size for individual pores formed by leishporin is around 32 nm, but indicate that they are prone to coalesce, originating large membrane damages that leads to cell collapse, what seems to be a unique property among pore-forming cytolysins.Author summaryOne of the mechanisms whereby a cell can be destroyed is by punching holes into their membranes. Through these holes, due to differences in osmolarity between the outside and the inside of a cell, water flows towards the cytoplasm causing plasma membrane ruptures, which damages or lyses cells. We have previously described in the protozoan parasite Leishmania amazonensis one of such activities. Using an electrophysiology technique, we have found that parasite extracts lyse cells by making pores on their membranes. However these pores were not directly visualized so far. In this report, using a high-resolution-type scanning microscopy, the atomic force microscopy, we showed in red blood cells membranes and artificial lipid membranes (liposomes) the physical aspect of the pores we described earlier. We observed that these pores are circular-shaped structures with variable diameters, ranging from 25 to 230 nm that span the whole thickness of both types of membranes. We verified that L. amazonensis extracts-mediated pores resemble to pores formed by lipids or peptides and not by pores formed by proteins and that they may fuse with one another forming larger holes.