Lysosomes are acidic organelles that are not only involved in degradation processes but also participated in other cellular functions, such as specialized secretion and plasma membrane (PM) resealing. When the PM is ruptured, Ca2+ flows from the extracellular milieu toward the cytoplasm potentially triggering cell death. In order to escape from the apoptotic route, cells developed an elegant mechanism in which lysosomes are recruited to the sites of injuries in a Ca2+-dependent fashion. Lysosomes, fuse with the PM releasing their enzymatic content. Acid sphingomyelinase (ASM), one of the secreted enzymes, cleaves sphingomyelin into ceramide, inducing compensatory endocytosis and internalization of the membrane-damaged site. Trypanosoma cruzi, the etiological agent of Chagas disease, relies heavily on lysosomes to successfully invade mammalian cells. By mechanically injuring the host PM, T. cruzi evokes lysosome exocytosis, and subsequently, compensatory endocytosis. The latter drives the parasite into the host cell, where it can replicate. This early association with lysosomes prevents T. cruzi evasion from the host cells allowing colonization of host intracellular milieu. This review chapter will summarize the main contributions in the field exploring the crosstalk between PM repair and T. cruzi invasion and how the understanding of these mechanisms evolved throughout the years.
- plasma membrane repair
- compensatory endocytosis
- Trypanosoma cruzi
The word lysosome is derived from the Greek words
Up to this date, more than 50 different enzymes were identified within lysosomes. Those membrane-delimited organelles are present in most nucleated mammalian cells. Lysosomes are mostly scattered across the cytoplasm but can become more concentrated around the perinuclear region upon stimuli . Lysosome intracellular movement is required for its proper functioning and has shown to be tightly regulated in the cell . Given their acidic interior, mostly composed by hydrolases, lysosomes are pivotal in intracellular degradation processes  such as intracellular digestion and autophagy [7, 8]. In order to digest endocytic cargo (membrane-bound vesicles resultant from pinocytosis or phagocytosis events) or autophagosomes, lysosomes have to fuse with those vesicles so their enzymes can have access to their content [9–11].
Besides being pivotal for intracellular degradation processes, lysosomes are also important for a plethora of physiological processes inside the cell, such as bone matrix resorption by osteoclasts , m-TOR-dependent antigen presentation by macrophages and dendritic cells , cholesterol transport , Ca2+-regulated PM resealing upon injury  and cell death , just to cite a few examples. Perturbations in lysosomal homeostasis, such as dysfunction of lysosomal hydrolases, impairment in lysosomal traffic and biogenesis might induce lysosomal storage disorders due to accumulation of unprocessed substrata inside this organelle. There are more than 50 different types of lysosomal storage diseases that were already identified .
As mentioned before, lysosomes play an important role in membrane resealing upon injury, and they are a fundamental part of the endocytic pathway. The endocytic pathway is basically composed by early and late endosomes and lysosomes. Internalized particles are delivered to early endosomes and are either recycled back to the membrane or transported to late endosomes. When they reach the late endosomes, the endocytosed material can be sorted by the Golgi apparatus and transported to the membrane or fuse with lysosomes to be degraded .
There is no doubt that the endocytic pathway is fundamental for nutrient uptake, cell signalling , and migration . A summary of the diverse cellular functions that the lysosomes are involved in is depicted in Figure 1. Intriguingly, the endocytic route is also explored by pathogens in order to successfully invade their host cells . Some of these pathogens evolved in order to develop mechanisms to evade lysosomal fusion in order to protect them from being degraded from lysosomal enzymes. However, in some cases, the pathogen drives itself to encounter lysosomes in order to guarantee intracellular survival. The gram-positive bacteria,
One of the most interesting pathogens that interact with lysosomes in order to successfully invade host cells is the protozoan parasite
2. From membrane resealing to
Trypanosoma cruziinvasion: what is the role played by lysosomes?
2.1. Plasma membrane injury and resealing: lysosomes save the day
It has been known since the early 90s that professional secretory cells, such as hepatocytes [41, 42], activated platelets [43, 44], pancreatic acinar cells [45, 46], macrophages [47, 48], osteoclasts [49, 50] and neutrophils [51, 52] are able to undergo regulated lysosomal secretion. However, until the mid-90s, it was not known whether non-professional secretory cells had the capability of performing lysosomal exocytosis. In 1995, Miyake and McNeil demonstrated for the first time that endothelial cells were able to accumulate vesicles near PM injured sites, and those vesicles underwent Ca2+ mediated exocytosis in order to seal those wounds . In 1996, Coorssen and colleagues have shown that epithelial cells enlarged their surface area by ~20–30% due to exocytosis promoted by increase in intracellular Ca2+. However, back then, they just hypothesized that the increase in area was probably due to secretion of endosomes or lysosomes . In 1997, Rodriguez and collaborators demonstrated that non-secretory cells, such as fibroblasts, myoblasts and epithelial cells, were able to trigger lysosomal exocytosis upon increase in intracellular Ca2+ levels. By performing enzymatic assays, they measured the presence of lysosomal enzymes, such as β-hexosaminidase and cathepsin D, in the supernatant of stimulated cells. In parallel, they also showed the presence of a lysosomal glycoprotein, Igp120, at the PM, corroborating the lysosomal exocytosis hypothesis .
Cells have evolved throughout time in order to develop a mechanism by which injuries in the PM could be quickly sealed in order to prevent cytoplasm leakage and cell death. Collagen matrix contraction assays for mimicking tissue morphogenesis and wound healing show that, upon contraction, fibroblasts can uptake extracellular dyes due to the formation of small pores in the membrane. Those small wounds are sealed within 5 s in the presence of Ca2+ . Tissues that are under mechanical stress, such as skeletal muscle , heart , gut  and skin  also have the ability to reseal their torn membranes and depend on this process for proper functioning. Impairment in sarcolemma resealing upon injury, for example, might cause muscular dystrophy .
2.2. Membrane resealing mechanism: from the patch hypothesis to acid sphingomyelinase-mediated compensatory endocytosis
The mechanism by which lysosomes reseal damaged plasma membranes was first proposed by Reddy and collaborators in 2001 . Using non-professional secretory cells, such as epithelial cells, myoblasts and fibroblasts, they showed that membrane injury upon scratching is able to trigger lysosomal exocytosis in a Ca2+-regulated manner. Similarly to neuronal synaptic vesicles that have a Ca2+-sensor protein called synaptotagmin I (syt-I) , lysosomes have an isoform of synaptotagmin named syt-VII [64, 65]. Synaptotagmins are proteins that have a short ectodomain (N terminus lumenal domain), a transmembrane region and two cytoplasmic domains C2A and C2B that are Ca2+-sensor domains. Reddy and colleagues demonstrated that the C2A domain is the one responsible for regulating Ca2+-dependent lysosomal exocytosis . Since then, it had been shown that lysosomes are able to undergo exocytosis in order to reseal PM injuries generated by different sources, such as pathogens  and pore-forming toxins , other than mechanical wounding. The most accepted model for PM repair in nucleated cells was proposed in the early 2000s and was called ‘The Patch Hypothesis’. According to that model, right underneath the injured site lysosomes underwent chaotic fusion events in which they either fused directly with the PM or with one another in a homotypical fusion manner. Those abnormally enlarged vesicles ended up fusing with the injured PM donating membrane to seal the wounded region [67, 68]. However, the patch model failed to explain the repair caused by pore-forming toxins, which stably binds to the membrane. Later, it was shown that the wounding caused by pore-forming toxins led to the formation of intracellular vesicles.
Wound healing experiments performed in the presence of gold-BSA, added prior to injury, demonstrated that those vesicles have an endocytic origin given that they retained gold-BSA in their lumen [15, 69]. Nonetheless, lysosomes play a pivotal role in the endocytosis-mediated plasma membrane resealing model. Following membrane lesion and increase in intracellular Ca2+, those organelles undergo exocytosis and secrete their enzymes into the extracellular medium. Acid sphingomyelinase (ASM) is one of the enzymes that remain active extracellularly after secretion, generating ceramide as a product of sphingomyelin hydrolysis [70, 71]. Ceramide coalesces at the membrane forming highly ordered domains excluding other lipids, such as glycerophospholipids, from those patches . Those domains induce membrane curvature and budding [71, 73, 74] dragging the injured region inward, in a processes called compensatory endocytosis, closing the wound. Cells either deficient in ASM or pharmacologically inhibited fail to undergo compensatory endocytosis but still trigger lysosomal exocytosis. Addition of recombinant ASM to the extracellular medium is able to restore compensatory endocytosis in those cells . Other lysosomal enzymes are also important to regulate the process. It has been proposed that cysteine proteases, cathepsins B and L, released during lysosomal exocytosis may contribute to facilitate ASM access to PM . Additionally, cathepsin D, another lysosomal enzyme released upon exocytosis, becomes active only later after its release and is responsible for negatively modulating ASM activity, closing the wounding cycle . Figure 2 depicts a timeline illustrating the evolution of the experimental models that explains how Ca2+-dependent membrane resealing upon lysosomal exocytosis is regulated within cells.
Trypanosoma cruzi: how this parasite can take advantage of intracellular endocytic route to perpetuate its intracellular cycle: the essential role of lysosomes in the process
Trypanosoma cruziand Chagas disease
During a blood meal, the insect excretes, together with the urine and faeces, the metacyclic trypomastigotes, which are capable of infecting the vertebrate host. These released trypomastigotes reach the mammalian host bloodstream either via the wound site or through mucous membranes. Once inside the vertebrate host the metacyclic trypomastigotes can infect a plethora of nucleated cells. When the parasite invades the host cell, it can differentiate into the amastigote form, which is the replicative form on the mammalian host. After several rounds of replication, the amastigotes differentiate into the trypomastigote form and the cells, crowded with parasites, burst open. Extracellular trypomastigotes are now free to perpetuate their cycle and infect new cells and tissues. The process that comprises from intracellular invasion to intracellular multiplication, and cell rupture takes about 4–5 days [79, 80].
Recent statistics provided by World Health Organization (WHO) website shows that about 6–7 million people are estimated to be infected with
Chagas disease has two phases: acute and chronic. The acute phase lasts from 4 to 8 weeks, and it is usually asymptomatic. However, mild symptoms like fever, for example, might happen 1–2 weeks after infection from the insect vector bite or a month later in other cases of transmission. Only 5–10% of the symptomatic cases might lead to death . The patients who survive from the acute phase will enter chronic phase, which lasts for the patient’s lifespan. The majority of the individuals that enter the chronic phase have the indeterminate form of the disease. However, 30–40% of the patients will potentially develop cardiomyopathy, 10% will develop megaesophagus, megacolon, cardiodigestive or neurological problems [37, 85, 86]. Until this day, there is no vaccine available or 100% effective cure for Chagas disease, especially if the disease is diagnosed during the chronic phase. There are two drugs, benznidazole and nifurtimox, which have proven to be effective for some cases during the acute phase. However, their use is limited due to low availability and severe side effects .
Trypanosoma cruzientry in host mammalian cells
Once the parasite gets in contact with host cells, the internalization odyssey takes place. Among them, Ca2+ signalling as well as lysosomal recruitment and fusion with the parasitophorous vacuole have been shown to be pivotal for a successful invasion [39, 40] Those two components are also fundamental for modulating PM repair in nucleated mammalian cells, as already described in Section 2.2. We are going to explore on the next subsection, how
Trypanosoma cruziand lysosomes: importance during parasite entry, maturation and intracellular multiplication
The first evidence showing that
Two years later, Tardieux and colleagues demonstrated that by exposing NRK cells either to trypomastigotes or to membranes isolated from trypomastigotes, Ca2+ transients were elicited in the host cell cytoplasm after only 200 s of exposure, which is faster than the invasion process
In 1995, two other papers from Dr. Norma Andrews’ group demonstrated that a Trypomastigote soluble peptidase (also referred to as Proteolytically Generated Trypomastigote Factor—PGTF) was able to generate Ca2+ transients in NRK cells . They also proved that PGTF is an agonist of PLC/IP3 generating Ca2+ transients, ultimately leading to actin cytoskeleton remodelling which facilitates
Years later, in 2001, Wilkowski and collaborators showed that incubation of phagocytic and non-professional phagocytic cells with phosphatidylinositol 3-kinase (PI3K) inhibitors, prior to
Two years later, Woolsey and collaborators demonstrated that even though lysosomes were important for
In 2004, Andrade and Andrews demonstrated that parasites that entered the host cell via PM-invagination mechanism gradually escape cells if they do not associate with lysosomal markers, demonstrating that association with lysosomes was pivotal for a successful invasion . Therefore, in the early 2000s, there were two convergent accepted models for
The fact that
Lysosome fusion with plasma membrane induced upon membrane injury is a tightly regulated process and dependent on PM cholesterol content . In 2012, Hissa and collaborators demonstrated that cholesterol depletion of cardiomyocytes prior to exposure to trypomastigotes changed the distribution of lysosomes within the host cell and evoked a massive lysosomal exocytosis near the cell cortex, even in the absence of extracellular Ca2+ . These critical lysosomal exocytic events led to a decrease in parasite internalization and lysosomal association for parasitophorous vacuole maturation . One year later, Hissa and colleagues proposed a mechanism by which cholesterol depletion triggered intracellular Ca2+-independent lysosomal secretion. Using methyl-beta cyclodextrin (MβCD) to chelate cholesterol from PM, they showed, by measuring mechanical properties of cell cortices, that cholesterol-depleted cells become more rigid with less membrane fluctuations . This work corroborated previous studies done in cholesterol-depleted endothelial cells . In line with that, cholesterol depletion induced Rho activation, which in turn led to actin polymerization enhancing cortical rigidity. Most importantly, the authors showed that lysosomal exocytosis triggered upon cholesterol depletion was not only Ca2+ but also Syt-VII independent, pointing out to a non-regulated secretion of those organelles. They suggested that actin polymerization induced by cholesterol depletion was responsible for the secretion of a lysosomal pool near the cell cortex. Based on these results, one can conclude that cells should have at least two different pools of these organelles, one located closer to the cell cortex, and most likely to be involved with membrane resealing events, and the second located closer to the cell nuclei and probably related to intracellular digestion. For the first pool, actin polymerization could work as an exocytic driving force, whereas for the second, it would present as a barrier for fusion with the PM. In fact, treatment of cells with Latrunculin-A, an actin filament-disrupting drug, induced the secretion of a more internally localized lysosomal pool . In 2015, Hissa and Andrade demonstrated that
Regarding intracellular development,
Lysosomal membrane proteins are also important for
As exposed here, opposite to other pathogens,
We would like to acknowledge the following Brazilian funding agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Instituto Nacional de Ciência de Tecnologia de Fluidos Complexos (INCT-FCx).