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The host’s ability to eradicate or control infection caused by intracellular pathogens depends on early interactions between these microorganisms and host cells. These events are related to the organism’s nature and stage of development and host immune status. Pathogens are recognized by host cells, which respond to infection by either mounting an efficient response or becoming a replication niche. Early interactions between the protozoan Leishmania parasite and host cell receptors activate different signaling pathways that can result in microbe elimination or, alternatively, infection establishment and the migration of Leishmania infected cells to other host tissues. This chapter focuses on Leishmania-macrophage interaction via phagocytosis, which involves a range of parasite ligands characteristic of Leishmania species and parasite stage of development and diverse host cell receptors. We also discuss alternative Leishmania entry by cell invasion and review how Leishmania spp. survive and replicate within the phagocytic compartment they induce.

The plasma membrane not only protects the cell from the extracellular environment, acting as a selective barrier, but also regulates cellular events that originate at the cell surface, playing a key role in various biological processes that are essential for the preservation of cell homeostasis. Therefore, elucidation of the mechanisms involved in the maintenance of plasma membrane integrity and functionality is of utmost importance. Cells have developed mechanisms to ensure the quality of proteins that inhabit the cell surface, as well as strategies to cope with injuries inflicted to the plasma membrane. Defects in these mechanisms can lead to the development or onset of several diseases. Despite the importance of these processes, a comprehensive and holistic perspective of plasma membrane quality control is still lacking. To tackle this gap, in this Review, we provide a thorough overview of the mechanisms underlying the identification and targeting of membrane proteins that are to be removed from the cell surface, as well as the membrane repair mechanisms triggered in both physiological and pathological conditions. A better understanding of the mechanisms underlying protein quality control at the plasma membrane can reveal promising and unanticipated targets for the development of innovative therapeutic approaches.

Although the genus Trichoderma is widely used as a biocontrol agent in crops, little is known about its potential impact on the human immune system. In mice, our group has shown that exposition to T. asperelloides spores lead to reduced neutrophil counts in the peripheral blood and in the peritoneal cavity. In addition, T. stromaticum spores produced an inflammatory infiltrate on mice lungs, reducing the levels of IFN-γ and IL-10 cytokines, reactive oxygen species, and receptors of microbial patterns. Here we demonstrate that the interaction of human peripheral neutrophils with T. stromaticum spores also leads to a reduced release of neutrophil extracellular traps (NETs) after induction with the NET-inducer agent phorbol 12-myristate 13-acetate. This interaction also reduced the expression levels of multiple microRNAs, such as miR-221, miR-222, miR-223 and miR-27a, as well as genes related to NETs, such as ELANE, MPO and PADI4. Furthermore, T. stromaticum spores affected the expression of the genes SOCS3, TLR4, CSNK2A1, GSDMD, and NFFKBIA, related to the activation of inflammatory immune responses in neutrophils. Overall, our results suggest T. stromaticum as a potential NET inhibitor and as an immunomodulatory agent. Since this fungus is used as biocontrol in crops, our findings point to the importance of advancing our knowledge on the effects of this bioagent on the human immune system. Finally, the study of the active compounds produced by the fungus is also important for the prospection of new drugs that could be used to block the exacerbation of inflammatory immune responses present in several human diseases.

Cell signalling, cell secretion, and plasma membrane repair are processes that critically rely on intracellular vesicles, important components of the endocytic and secretory pathways. More specifically, the strategic distribution of intracellular vesicles is important for diverse cellular processes. The method presented here is a simple, affordable, and efficient tool to analyze the distribution of intracellular vesicles such as lysosomes, endosomes, Golgi vesicles or secretory granules under different experimental conditions. The method is an accessible way to analyze the density and dispersion of intracellular vesicles by combining immunofluorescence with pixel-based quantification software (e.g., ImageJ/FIJI). This protocol can be used widely within the scientific community because it utilizes ImageJ/FIJI, an open source software that is free. By tracking fluorescent vesicles based on their position relative to cell nuclei we are able to quantify and analyze their distribution throughout the cell.

Leishmania (Viannia) guyanensisis one species that causes cutaneous leishmaniasis in the New World. The incidence of infections with this parasite is probably underestimated and few studies exist on this species, despite its epidemiological importance. In particular, there are no studies concerningL. guyanensismetacyclogenesis and no technique for obtaining metacyclic promastigotes for this species is presently available. Here, we have studiedL. guyanensismetacyclogenesis in axenic culture, describing the main changes that occur during this process, namely, in morphology and size, sensitivity to complement-mediated lysis, surface carbohydrates and infectivity to macrophages. We have shown that metacyclogenesis inL. guyanensispromastigotes is basically complete on the 4thday of culture, as determined by decreased body size, increased flagellum length, resistance to complement-mediated lysis and infectivity. We have also found that only a fraction of the parasites is agglutinated byBauhinia purpurealectin. The non-agglutinated parasites, which also peaked on the 4thday of culture, had all morphological traits typical of the metacyclic stage. This is the first report describing metacyclogenesis inL. guyanensisaxenic promastigotes and a simple and efficient method for the purification of metacyclic forms. Furthermore, a model of human macrophage infection withL. guyanensiswas established.

Intracellular parasites from the genera Toxoplasma, Plasmodium, Trypanosoma, Leishmania and from the phylum Microsporidia are, respectively, the causative agents of toxoplasmosis, malaria, Chagas disease, leishmaniasis and microsporidiosis, illnesses that kill millions of people around the globe. Crossing the host cell plasma membrane (PM) is an obstacle these parasites must overcome to establish themselves intracellularly and so cause diseases. The mechanisms of cell invasion are quite diverse and include (1) formation of moving junctions that drive parasites into host cells, as for the protozoans Toxoplasma gondii and Plasmodium spp., (2) subversion of endocytic pathways used by the host cell to repair PM, as for Trypanosoma cruzi and Leishmania, (3) induction of phagocytosis as for Leishmania or (4) endocytosis of parasites induced by specialized structures, such as the polar tubes present in microsporidian species. Understanding the early steps of cell entry is essential for the development of vaccines and drugs for the prevention or treatment of these diseases, and thus enormous research efforts have been made to unveil their underlying biological mechanisms. This Review will focus on these mechanisms and the factors involved, with an emphasis on the recent insights into the cell biology of invasion by these pathogens.

The ability of repairing damages on the plasma membrane is crucial for cell survival. When damaged, eukaryotic cells are able to recover plasma membrane integrity within a few seconds, thus avoiding cytoplasm leakage and cell death. The process is driven by the influx of extracellular calcium which triggers a multitude of intracellular effects that participate in the process of plasma membrane resealing. One of the landmarks of plasma membrane repair is the triggering of intracellular vesicles recruitment and their exocytosis at damage sites. Since lysosomes are able to respond to calcium influx and that some of the lysosomal enzymes exocytosed after plasma membrane permeabilization are essential to restore cell integrity, these organelles have emerged as essential for the maintenance of plasma membrane integrity. Here we summarize the scientific evidences showing the involvement of lysosomes in plasma membrane repair that allowed researchers to propose a totally different function for this famous organelle.

Leishmaniasis is a disease caused by trypanosomatid protozoa of the genus Leishmania. In the Americas, the species Leishmania amazonensis is predominantly associated with American cutaneous leishmaniasis (ACL) while L. infantum is an agent of visceral leishmaniasis (VL). The genome sequences of Leishmania spp. have shown that each genome can contain about 8000 genes encoding proteins, more than half of which have an unknown function (''hypotheticals") at the time of publication. To understand the biology and genome of the organisms, it is important to discover the function of these "hypothetical" proteins; however, few studies have focused on their characterizations. Previously, LinJ.30.3360 (a protein with unknown function) was identified as immunogenic to canine serum with VL and a good antigen to diagnose the visceral form in dogs. Here, we show that the LinJ.30.3360 protein is conserved in L. infantum, L. tarantolae, L. donovani, L. major, L. mexicana, L. braziliensis, L. panamensis, Leptomonas pyrrhocoris, and Leptomonas seymouri. It has been annotated as a MORN (Membrane Occupation and Recognition Nexus) domain protein. However, since the function of this motif is unknown, functional inferences based on the primary sequence are not possible. The protein has a folded β-leaf secondary structure, and phosphorylation was the only post-translational modification (PTM) found using prediction approach. Experiments have shown that it is located close to the flagellar pocket and presents similar abundance in both L. amazonensis and L. infantum. Furthermore, because it is a conserved protein in trypanosomatids but not in mammals and also because of its antigenicity, LinJ.30.3360 may constitute a potential drug target and/or vaccine for leishmaniasis.

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.

The ability of repairing damages on the plasma membrane is crucial for cell survival. When damaged, eukaryotic cells are able to recover plasma membrane integrity within a few seconds, thus avoiding cytoplasm leakage and cell death. The process is driven by the influx of extracellular calcium which triggers a multitude of intracellular effects that participate in the process of plasma membrane resealing. One of the landmarks of plasma membrane repair is the triggering of intracellular vesicles recruitment and their exocytosis at damage sites. Since lysosomes are able to respond to calcium influx and that some of the lysosomal enzymes exocytosed after plasma membrane permeabilization are essential to restore cell integrity, these organelles have emerged as essential for the maintenance of plasma membrane integrity. Here we summarize the scientific evidences showing the involvement of lysosomes in plasma membrane repair that allowed researchers to propose a totally different function for this famous organelle.

The purpose of this study was to investigate the role of pentraxin 3 (PTX3), a pivotal component of the innate immune system, in gout. Levels of PTX3 and IL-1β in human samples were evaluated by ELISA. Development of murine gout was evaluated through the levels of cytokines (PTX3, CXCL1, and IL-1β) and neutrophil recruitment into the joint cavity. Phagocytosis of monosodium urate (MSU) crystals and caspase-1 activation were determined by flow cytometer. Acute gout patients showed elevated concentration of PTX3 in plasma and synovial fluid as compared with healthy and osteoarthritic subjects. Moreover, there was a positive correlation between intra-articular PTX3 and IL-1β levels. PTX3 was induced in the periarticular tissue of mice postinjection of MSU crystals. Importantly, Ptx3-deficient mice showed reduced inflammation in response to MSU crystal injection compared with wild-type mice, including reduction of neutrophil recruitment into the joint cavity and IL-1β and CXCL1 production. Interestingly, addition of PTX3 in vitro enhanced MSU crystal phagocytosis by monocytes and resulted in higher production of IL-1β by macrophages. This contribution of PTX3 to the phagocytosis of MSU crystals and consequent production of IL-1β occurred through a mechanism mainly dependent on FcγRIII. Thus, our results suggest that PTX3 acts as a humoral pattern recognition molecule in gout facilitating MSU crystal phagocytosis and contributing to the pathogenesis of gouty arthritis.

Intracellular 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, is that other cell types and even non-phagocytic cells have been found to be infected by Leishmania spp. Nevertheless, the mechanisms by which Leishmania invades such cells had not been previously studied. Here, we show that L. amazonensis can induce their own entry into fibroblasts independently of actin cytoskeleton activity, and, thus, through a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions, such as Ca2+ signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair. This article has an associated First Person interview with the first author of the paper.

Plasma membrane repair (PMR) is an important process for cell homeostasis, especially for cells under constant physical stress. Repair involves a sequence of Ca2+-dependent events, including lysosomal exocytosis and subsequent compensatory endocytosis. Cholesterol sequestration from plasma membrane causes actin cytoskeleton reorganization and polymerization, increasing cell stiffness, which leads to exocytosis and reduction of a peripheral pool of lysosomes involved in PMR. These changes in mechanical properties are similar to those observed in cells exposed to oxidized Low Density Lipoprotein (oxLDL), a key molecule during atherosclerosis development. Using a human umbilical vein endothelial cell line (EAhY926) we evaluated the influence of mechanical modulation induced by oxLDL in PMR and its effect in endothelial fragility. Similar to MβCD (a drug capable of sequestering cholesterol) treatment, oxLDL exposure led to actin reorganization and de novo polymerization, as well as an increase in cell rigidity and lysosomal exocytosis. Additionally, for both MβCD and oxLDL treated cells, there was an initial increase in endocytic events, likely triggered by the peak of exocytosis induced by both treatments. However, no further endocytic events were observed, suggesting that constitutive endocytosis is blocked upon treatment and that the reorganized cytoskeleton function as a mechanical barrier to membrane traffic. Finally, the increase in cell rigidity renders cells more prone to mechanical injury. Together, these data show that mechanical modulation induced by oxLDL exposure not only alters membrane traffic in cells, but also makes them more susceptible to mechanical injury, which may likely contribute to the initial steps of atherosclerosis development.

Leishmania (Viannia) guyanensis is one species that causes cutaneous leishmaniasis in the New World. The incidence of infections with this parasite is probably underestimated and few studies exist on this species, despite its epidemiological importance. In particular, there are no studies concerning L. guyanensis metacyclogenesis and no technique for obtaining metacyclic promastigotes for this species is presently available. Here, we have studied L. guyanensis metacyclogenesis in axenic culture, describing the main changes that occur during this process, namely, in morphology and size, sensitivity to complement-mediated lysis, surface carbohydrates and infectivity to macrophages. We have shown that metacyclogenesis in L. guyanensis promastigotes is basically complete on the 4th day of culture, as determined by decreased body size, increased flagellum length, resistance to complement-mediated lysis and infectivity. We have also found that only a fraction of the parasites is agglutinated by Bauhinia purpurea lectin. The non-agglutinated parasites, which also peaked on the 4th day of culture, had all morphological traits typical of the metacyclic stage. This is the first report describing metacyclogenesis in L. guyanensis axenic promastigotes and a simple and efficient method for the purification of metacyclic forms. Furthermore, a model of human macrophage infection with L. guyanensis was established.

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.AUTHOR 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.

Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.

Gaucher disease (GD) is caused by bi-allelic mutations in GBA1, the gene that encodes acid β-glucocerebrosidase (GCase). Individuals affected by GD have hematologic, visceral and bone abnormalities, and in severe cases there is also neurodegeneration. To shed light on the mechanisms by which mutant GBA1 causes bone disease, we examined the ability of human induced pluripotent stem cells (iPSC) derived from patients with Types 1, 2 and 3 GD, to differentiate to osteoblasts and carry out bone deposition. Differentiation of GD iPSC to osteoblasts revealed that these cells had developmental defects and lysosomal abnormalities that interfered with bone matrix deposition. Compared with controls, GD iPSC-derived osteoblasts exhibited reduced expression of osteoblast differentiation markers, and bone matrix protein and mineral deposition were defective. Concomitantly, canonical Wnt/β catenin signaling in the mutant osteoblasts was downregulated, whereas pharmacological Wnt activation with the GSK3β inhibitor CHIR99021 rescued GD osteoblast differentiation and bone matrix deposition. Importantly, incubation with recombinant GCase (rGCase) rescued the differentiation and bone-forming ability of GD osteoblasts, demonstrating that the abnormal GD phenotype was caused by GCase deficiency. GD osteoblasts were also defective in their ability to carry out Ca(2+)-dependent exocytosis, a lysosomal function that is necessary for bone matrix deposition. We conclude that normal GCase enzymatic activity is required for the differentiation and bone-forming activity of osteoblasts. Furthermore, the rescue of bone matrix deposition by pharmacological activation of Wnt/β catenin in GD osteoblasts uncovers a new therapeutic target for the treatment of bone abnormalities in GD.

Repair of plasma membrane wounds in B lymphocytes that lack caveolin requires lysosome exocytosis and lipid raft–mediated endocytosis and inhibits activation of the B cell receptor by sequestering lipid rafts., Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca2+-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca2+-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.

Trypanosoma cruzi enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of T. cruzi into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2-/- cells, indicating that LAMP-2 is an important player during T. cruzi invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2-/- and LAMP1/2-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and T. cruzi ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in T. cruzi invasion., Author summary Trypanosoma cruzi is the etiological agent of Chagas disease, a very debilitating illness that has no efficient treatment to date. Better knowledge of the mechanisms involved with host cell infection is important to change this scenario. T. cruzi enters host cells by subverting the mechanism by which cells repair small injuries in their plasma membrane. In this process, parasites interact with host cells causing membrane injuries. These injuries lead to secretion of lysosomal content to the extracellular media, which in turn causes the internalization of plasma membrane wounds via endocytosis. During this endocytic process the parasite is internalized by the host cell. We have previously shown that absence of two proteins located at the lysosomal membrane, LAMP-1 and 2, severely interferes with T. cruzi entry into host cells. In the present manuscript we show that absence of LAMP-2 alone is enough to compromise T. cruzi entry into host cells and it does so by compromising the endocytic events that follow lysosome secretion, which in turn is responsible for driving T. cruzi into the host cell interior.

The pathogenesis of malaria is complex, generating a broad spectrum of clinical manifestations. One of the major complications and concerns in malaria is anemia, which is responsible for considerable morbidity in the developing world, especially in children and pregnant women. Despite its enormous health importance, the immunological mechanisms involved in malaria-induced anemia remain incompletely understood. Plasmodium vivax , one of the causative agents of human malaria, is known to induce a strong inflammatory response with a robust production of immune effectors, including cytokines and antibodies. Therefore, it is possible that the extent of the immune response not only may facilitate the parasite killing but also may provoke severe illness, including anemia. In this review, we consider potential immune effectors and their possible involvement in generating this clinical outcome during P. vivax infections.

We have previously shown that various species of Leishmania produce a lytic activity, which, in Leishmania amazonensis, is mediated by a pore-forming cytolysin, called leishporin. It is toxic for macrophages in vitro and optimally active at pH 5.0 to 5.5 and at 37 °C, suggesting that it might be active inside phagolysosomes. Leishporin from both L. amazonensis (a-leishporin) and Leishmania guyanensis (g-leishporin) can be activated by proteases, suggesting either a limited proteolysis of an inactive precursor or a proteolytic degradation of a non-covalently linked inhibitor. Here, we show that both a- and g-leishporin are also activated in dissociating conditions, indicating the second hypothesis as the correct one. In fact, we further demonstrated that inactive leishporin is non-covalently associated with an inhibitor, possibly more than one oligopeptide that, when removed, renders leishporin hemolytically active. This activation was shown to be the result of both the improvement of leishporin's ability to bind to phospholipids and the emergence of its pore-forming ability. In vitro results demonstrate that leishporin can be released by the parasites, as they evolve in axenic cultures, in an inactive form that can be activated. These results are compatible with our hypothesis that leishporin can be activated in the protease-rich, low pH, and dissociating environment of parasitophorous vacuoles, leading to disruption of both vacuoles and plasma membranes with the release of amastigotes.

Mutations of the Plekhm1 gene in humans and rats cause osteopetrosis, an inherited bone disease characterized by diminished bone resorption by osteoclasts. PLEKHM1 binds to RAB7 and is critical for lysosome trafficking. However, the molecular mechanisms by which PLEKHM1 regulates lysosomal pathways remain unknown. Here, we generated germline and conditional Plekhm1-deficient mice. These mice displayed no overt abnormalities in major organs, except for an increase in trabecular bone mass. Furthermore, loss of PLEKHM1 abrogated the peripheral distribution of lysosomes and bone resorption in osteoclasts. Mechanistically, we indicated that DEF8 interacts with PLEKHM1 and promotes its binding to RAB7, whereas the binding of FAM98A and NDEL1 with PLEKHM1 connects lysosomes to microtubules. Importantly, suppression of these proteins results in lysosome positioning and bone resorption defects similar to those of Plekhm1-null osteoclasts. Thus, PLHKEM1, DEF8, FAM98A, and NDEL1 constitute a molecular complex that regulates lysosome positioning and secretion through RAB7.

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.

The study of plasma membrane repair is coming of age. Mirroring human adolescence, the field shows at the same time signs of maturity and significant uncertainty, confusion and skepticism. Here we discuss concepts that emerged from experimental data over the years, some of which are solidly established while others are still subject to different interpretations. The firmly established concepts include the critical requirement for Ca(2+) in wound repair, and the role of rapid exocytosis of intracellular vesicles. Lysosomes are being increasingly recognized as the major vesicles involved in injury-induced exocytosis in many cell types, as a growing number of laboratories detect markers for these organelles on the cell surface and lysosomal hydrolases in the supernatant of wounded cells. The more recent observation of massive endocytosis following Ca(2+)-triggered exocytosis initially came as a surprise, but this finding is also being increasingly reported by different groups, shifting the discussion to the mechanisms by which endocytosis promotes repair, and whether it operates or not in parallel with the shedding of membrane blebs. We discuss how the abundant intracellular vesicles that undergo homotypic fusion close to wound sites, previously interpreted as exocytic membrane patches, actually acquire extracellular tracers demonstrating their endocytic origin. We also suggest that an initial, temporary patch that prevents cytosol loss until the bilayer is restored might result not from vesicular fusion, but from rapid Ca(2+)-dependent crosslinking and aggregation of cytosolic proteins. Finally, we propose that cell surface remodeling, orchestrated by the extracellular release of lysosomal hydrolases and perhaps also cytosolic molecules, may represent a key aspect of the plasma membrane repair mechanism that has received little attention so far.

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.

Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane.

Rapid repair of plasma membrane wounds is critical for cellular survival. Exocytic patches, membrane tension reduction and endocytosis were previously proposed to mediate resealing. A recent study implicating the ESCRT complex adds to the growing evidence that repair involves removal of damaged plasma membrane, and not simply patching the wound.

Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca2+-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins. DOI: http://dx.doi.org/10.7554/eLife.00926.001, eLife digest Cells must be able to rapidly repair damage to their outer membranes. This is particularly important in the case of muscle cells, which are vulnerable to damage, and the failure of these cells to repair their outer membranes leads to the muscle wastage seen in muscular dystrophy. Researchers do not fully understand how cells repair membrane, but one popular theory is that they use the membranes of specialized vesicles to ‘patch’ areas that have been damaged. A group of proteins called caveolins have also been implicated in membrane repair but, again, the details have not been worked out. These proteins are best known for their role in the formation of caveolae — small pouches formed by invaginated sections of the plasma membrane. Now, Corrotte et al. have obtained evidence that membrane repair relies not on patching, but on endocytosis (the process by which substances are taken into the cell in small vesicles that ‘pinch’ from the plasma membrane) of these caveolae pouches. Corrotte et al. treated cells with streptolysin O, a toxin that forms pores in the membrane that cannot be repaired using patches, and found that this led to the formation of small membrane-derived vesicles that looked just like caveolae. Further tests confirmed that these vesicles contained caveolar proteins, and that they removed the toxin from the plasma membrane by endocytosis. Similar effects were seen in response to mechanical damage caused by tiny glass beads. Moreover, blocking the expression of caveolar genes prevented cells from repairing membrane damage. Based on their findings, Corrotte et al. propose an alternative model for the repair process; namely that cellular damage triggers an influx of calcium ions, which causes vesicles called lysosomes to release chemicals that promote the formation of caveolae. These then remove the damaged area through endocytosis, restoring the integrity of the membrane. The results offer new insights into why mutations in caveolar proteins are associated with muscle disorders, including muscular dystrophy and cardiac dysfunction. DOI: http://dx.doi.org/10.7554/eLife.00926.002

Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.

To lyse cells, some pore-forming proteins need to bind to receptors on their targets. Studying the binding requirements of Leishmania amazonensis leishporin, we have shown that protease-treated erythrocytes are as sensitive to leishporin-mediated lysis as untreated cells, indicating that protein receptors are dispensable. Similarly, carbohydrate receptors do not seem to be needed, since several sugars do not inhibit leishporin-mediated hemolysis. Conversely, dipalmitoylphosphatidylcholine (DPPC), but not cholesterol, completely inhibits leishporin-mediated lysis. DPPC liposomes, with or without cholesterol, are lysed by leishporin and remove its lytic activity. Our results demonstrate that leishporin is a cholesterol-independent cytolysin that binds directly to phospholipids.

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The plasma membrane of B cells is at risk for injury by bacterial toxins, viruses, and possible mechanical damage induced by interactions with other cells during antigen presentation. Whether and how B cells repair membrane damage and its impact on B-cell functions are unknown. Here we show that upon injury by the pore forming toxin streptolysin O (SLO), primary mouse splenic B cells can repair their membrane in a Ca2+-dependent manner, like muscle and epithelial cells. B cells exocytose their lysosomes in response to Ca2+ influx and release acid sphingomyelinase from lysosomes, which induces endocytosis. In B cells activated by cross-linking B-cell receptors (BCRs), repairing membrane damage by SLO inhibits BCR clustering and internalization, consequently reducing BCR-induced tyrosine phosphorylation. Further analyses show that membrane repair inhibits the colocalization of BCRs with lipid rafts on the cell surface and during their internalization, indicating that membrane repair-induced endocytosis competes with BCRs for lipid rafts and interferes with the interaction of BCRs with lipid raft-associated kinases. Our results reveal the ability and the mechanism for B cells to repair membrane damage, and discover a negative regulatory role of membrane repair on B cell signaling and antigen uptake functions.