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

Abstract

26We have previously shown that Leishmania amazonensis produces and secretes a 27 cytolysin that lyses membranes of mammalian cells, including macrophages, its host 28 cell. Using the patch-clamp technique, we have previously demonstrated that the 29 mechanism by which this cytolysin rupture macrophages plasma membrane is by pore 30 formation, which lead us to name it leishporin. While we have characterized leishporin 31 in several aspects, its molecular identity is still unknown. Its behavior suggests that 32 leishporin is, or depend on, a protein, but recent results also suggests that a non-protein 33 molecule is involved in cell lysis. Although the patch-clamp has undeniably revealed 34 that L. amazonensis extracts generates pores in macrophages, these structures have not 35 been spotted on cell membranes, which prompted us to several questions: 1) What is the 36 appearance of leishporin-induced pores? Is it similar to that of other described pores? 2) 37 Do these pores physically span lipid bilayers? 4) Are their directly-measured sizes 38 compatible with those previously suggested by patch-clamp? 5) Do these pores fuse 39 with one another, enlarging in size, as suggested by our previous reports? In the present 40 work, we have used two membrane models, erythrocytes and liposomes, to visualize 41 pores induced by the cytolysin on parasite extracts. Leishporin-mediated lysed 42 erythrocytes or liposomes were analyzed by atomic force microscopy (AFM), which 43 allowed us to visualize multiple membrane-spanning pores of variable diameters, 44 ranging from 25 to 230 nm. They do not resemble to protein-formed pores, but rather, to 45 pores made by small molecules such as lipids or peptides, as also visualized by AFM. 46 Our results suggest that the maximum size for individual pores formed by leishporin is 47 around 32 nm, but indicate that they are prone to coalesce, originating large membrane 48 damages that leads to cell collapse, what seems to be a unique property among pore-49 forming cytolysins. 3 50Author summary 51 One of the mechanisms whereby a cell can be destroyed is by punching holes into their 52 membranes. Through these holes, due to differences in osmolarity between the outside 53 and the inside of a cell, water flows towards the cytoplasm causing plasma membrane 54 ruptures, which damages or lyses cells. We have previously described in the protozoan 55 parasite Leishmania amazonensis one of such activities. Using an electrophysiology 56 technique, we have found that parasite extracts lyse cells by making pores on their 57 membranes. However these pores were not directly visualized so far. In this report, 58 using a high-resolution-type scanning microscopy, the atomic force microscopy, we 59 showed in red blood cells membranes and artificial lipid membranes (liposomes) the 60 physical aspect of the pores we described earlier. We observed that these pores are 61 circular-shaped structures with variable diameters, ranging from 25 to 230 nm that span 62 the whole thickness of both types of membra...