Biology subjects

Horta, M. F.

Publications and source records attributed to Horta, M. F..

3 recordsLinked to original sources

bioRxiv2025

Leishmania amazonensis amastigotes invade non-phagocytic cells via highly localized parasite-induced actin remodeling.

Intracellular parasites are pathogens that must invade and persist within host cells. In the case of Leishmania spp., it is generally assumed that the parasite must be phagocytosed to further establish residence within professional phagocytic cells. However, several studies report the presence of Leishmania spp. in non-phagocytic cells, highlighting their ability to enter cells independently of classical phagocytosis. Indeed, we have recently demonstrated that promastigotes, the infective form transmitted to hosts during the bite of the insect vector, subvert a lysosome-exocytosis-dependent membrane repair pathway to invade fibroblasts. Here, we investigate the invasion of non-phagocytic cells by L. amazonensis amastigotes, the infective form directly responsible for host-to-vector transmission, disease generation and parasite dissemination during infection in humans and other mammalian hosts. Our results show that amastigotes rapidly induce their entry into cells lacking classical phagocytic capability, where they survive, multiply, and persist. Invasion depends on parasite-induced actin remodeling confined to the parasite-host plasma membrane contact site, with localized recruitment of Rho GTPases that fuel actin polymerization at invasion foci. Our findings highlight the remarkable ability of Leishmania amastigotes to induce their own internalization into virtually any cell type, bypassing the need for conventional phagocytosis. This property may profoundly influence parasite biology by enabling amastigotes to cross cellular barriers, disseminate, and silently establish infection in diverse host cells. Importantly, when considering non-phagocytic cells, our results demonstrate that Leishmania amazonensis employs distinct, stage-specific invasion mechanisms: promastigotes co-opt host cell lysosomes, whereas amastigotes depend on F-actin dynamics. SUMMARYHere we elucidate key aspects of the mechanism by which Leishmania amazonensis amastigotes invade non-phagocytic cells.

microbiology↗

bioRxiv2019

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.\n\nAuthor 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.

microbiology↗

bioRxiv2018

Unveiling Leishmania invasion of fibroblasts: calcium signaling, lysosome recruitment and exocytosis culminate with actin-independent invasion.

Intracellular parasites of the genus Leishmania are the causative agents of human leishmaniasis, a widespread emergent tropical disease. The parasite is transmitted by the bite of a hematophagous sandfly vector that inoculates motile flagellated promastigote forms into the dermis of the mammalian host. After inoculation, parasites are ultimately captured by macrophages and multiply as round-shaped amastigote forms. Macrophages seem not to be the first infected cells since parasites were observed invading neutrophils first whose leishmania-containing apoptotic bodies were latter captured by macrophages, thereby becoming infected. The fact that Leishmania spp are able to live and replicate inside immune phagocytic cells and that macrophages are the main cell type found infected in chronicity created the perception that Leishmania spp are passive players waiting to be captured by phagocytes. However, several groups have described the infection of non-phagocytic cells in vivo and in vitro. The objective of this work was to study the cellular mechanisms involved in the invasion of non-professional phagocytes by Leishmania. We show that promastigotes of L.amazonensis actively induces invasion in fibroblasts without cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Inside fibroblasts parasites transformed in amastigotes, remained viable for at least two weeks and re-transformed in promastigotes when returned to insect vector conditions. Similarly to what was observed for T. cruzi, infection involves calcium signaling, recruitment and exocytosis of lysosomes involved in plasma membrane repair and lysosome-triggered endocytosis. Conditions that alter lysosomal function such as cytochalasin-D and brefeldin-A treatment or the knockout of host cell lysosomal proteins LAMP-1 and 2 dramatically affected invasion. Likewise, triggering of lysosomal exocytosis and lysosome-dependent plasma membrane repair by low doses of streptolysin-O dramatically increased parasite entry. Together our results show that L.amazonensis promastigotes are able to take advantage of calcium-dependent lysosomal exocytosis and lysosome-induced endocytosis to invade and persist in non-phagocytic cells.\n\nAUTHOR SUMMARYIntracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting but overlooked finding on Leishmania infection is that non-phagocytic cells have also been found infected by amastigotes. Nevertheless, the mechanisms by which Leishmania invades non-phagocytic cells were not studied to date. Here we show that L. amazonensis can actively induce their own entry into fibroblasts independently of actin cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions such as calcium signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair and whose positioning and content interfere in invasion. Parasites were able to replicate and remained viable in fibroblasts, suggesting that cell invasion trough the mechanism demonstrated here could serve as a parasite hideout and reservoir, facilitating infection amplification and persistence.

cell biology↗