mTOR signaling inhibition decreases lysosome migration and impairs the success of Trypanosoma cruzi infection and replication in cardiomyocytes.

Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi) and, among all the chronic manifestations of the disease, Chronic Chagas Cardiomyopathy (CCC) is the most severe outcome. Despite high burden and public health importance in Latin America, there is a gap in understanding the molecular mechanisms that results in CCC development. Previous studies showed that T. cruzi uses the host machinery for infection and replication, including the repurposing of the responses to intracellular infection such as mitochondrial activity, vacuolar membrane, and lysosomal activation in benefit of parasite infection and replication. One common signaling upstream to many responses to parasite infection is mTOR pathway, previous associated to several downstream cellular mechanisms including autophagy, mitophagy and lysosomal activation. Here, using human iPSC derived cardiomyocytes (hiPSCCM), we show the mTOR pathway is activated in hiPSCCM after T. cruzi infection, and the inhibition of mTOR with rapamycin reduced number of T. cruzi 48 h post infection (hpi). Rapamycin treatment also reduced lysosome migration from nuclei region to cell periphery resulting in less T. cruzi inside the parasitophorous vacuole (PV) in the first hour of infection. In addition, the number of parasites leaving the PV to the cytoplasm to replicate in later times of infection was also lower after rapamycin treatment. Altogether, our data suggest that host's mTOR activation concomitant with parasite infection modulates lysosome migration and that T. cruzi uses this mechanism to achieve infection and replication. Modulating this mechanism with rapamycin impaired the success of T. cruzi life cycle independent of mitophagy.

Successful invasion of Trypanosoma cruzi trypomastigotes is dependent on host cell actin cytoskeleton.

Cellular invasion by Trypanosoma cruzi metacyclic trypomastigotes (MTs) or tissue culture trypomastigotes (TCTs) is a complex process involving host-parasite cellular and molecular interactions. Particularly, the involvement of host cell actin cytoskeleton during trypomastigote invasion is poorly investigated, and still, the results are controversial. In the present work, we compare side by side both trypomastigote forms and employ state-of-the-art live-cell imaging showing for the first time the dynamic mobilization of host cell actin cytoskeleton to MT and TCT invasion sites. Moreover, cytochalasin D, latrunculin B, and jasplakinolide-pretreated cells inhibited MT and TCT invasion. Furthermore, our results demonstrated that TCT invasion decreased in RhoA, Rac1, and Cdc-42 GTPase-depleted cells, whereas MT invasion decreased only in Cdc42-and RhoA-depleted cells. Interestingly, depletion of the three studied GTPases induced a scattered lysosomal distribution throughout the cytosol. These observations indicate that GTPase depletion is sufficient to impair parasite invasion despite the importance of lysosome spread in trypomastigote invasion. Together, our results demonstrate that the host cell actin cytoskeleton plays a direct role during TCT and MT invasion.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

Guanylate Binding Proteins Restrict Leishmania donovani Growth in Nonphagocytic Cells Independent of Parasitophorous Vacuolar Targeting.

Interferon (IFN)-inducible guanylate binding proteins (GBPs) play important roles in host defense against many intracellular pathogens that reside within pathogen-containing vacuoles (PVs). For instance, members of the GBP family translocate to PVs occupied by the protozoan pathogen Toxoplasma and facilitate PV disruption and lytic parasite killing. While the GBP defense program targeting Toxoplasma has been studied in some detail, the role of GBPs in host defense to other protozoan pathogens is poorly characterized. Here, we report a critical role for both mouse and human GBPs in the cell-autonomous immune response against the vector-borne parasite Leishmania donovani Although L. donovani can infect both phagocytic and nonphagocytic cells, it predominantly replicates inside professional phagocytes. The underlying basis for this cell type tropism is unclear. Here, we demonstrate that GBPs restrict growth of L. donovani in both mouse and human nonphagocytic cells. GBP-mediated restriction of L. donovani replication occurs via a noncanonical pathway that operates independent of detectable translocation of GBPs to L. donovan-containing vacuoles (LCVs). Instead of promoting the lytic destruction of PVs, as reported for GBP-mediated killing of Toxoplasma in phagocytic cells, GBPs facilitate the delivery of L. donovani into autolysosomal-marker-positive compartments in mouse embryonic fibroblasts as well as the human epithelial cell line A549. Together our results show that GBPs control a novel cell-autonomous host defense program, which renders nonphagocytic cells nonpermissible for efficient Leishmania replication.IMPORTANCE The obligate intracellular parasite Leishmania causes the disease leishmaniasis, which is transmitted to mammalian hosts, including humans, via the sandfly vector. Following the bite-induced breach of the skin barrier, Leishmania is known to live and replicate predominantly inside professional phagocytes. Although Leishmania is also able to infect nonphagocytic cells, nonphagocytic cells support limited parasitic replication for unknown reasons. In this study, we show that nonphagocytic cells possess an intrinsic property to restrict Leishmania growth. Our study defines a novel role for a family of host defense proteins, the guanylate binding proteins (GBPs), in antileishmanial immunity. Mechanistically, our data indicate that GBPs facilitate the delivery of Leishmania into antimicrobial autolysosomes, thereby enhancing parasite clearance in nonphagocytic cells. We propose that this GBP-dependent host defense program makes nonphagocytic cells an inhospitable host cell type for Leishmania growth.

Lysosome assembly and disassembly changes endocytosis rate through the Leishmania cell cycle.

The Leishmania lysosome has an atypical structure, consisting of an elongated vesicle-filled tubule running along the anterior-posterior axis of the cell, which is termed the multivesicular tubule (MVT) lysosome. Alongside, the MVT lysosome is one or more microtubules, the lysosomal microtubule(s). Previous work indicated there were cell cycle-related changes in MVT lysosome organization; however, these only provided snapshots and did not connect the changes in the lysosomal microtubule(s) or lysosomal function. Using mNeonGreen tagged cysteine peptidase A and SPEF1 as markers of the MVT lysosome and lysosomal microtubule(s), we examined the dynamics of these structures through the cell cycle. Both the MVT lysosome and lysosomal microtubule(s) elongated at the beginning of the cell cycle before plateauing and then disassembling in late G2 before cytokinesis. Moreover, the endocytic rate in cells where the MVT lysosome and lysosomal microtubule(s) had disassembled was extremely low. The dynamic nature of the MVT lysosome and lysosomal microtubule(s) parallels that of the Trypanosoma cruzi cytostome/cytopharynx, which also has a similar membrane tubule structure with associated microtubules. As the cytostome/cytopharynx is an ancestral feature of the kinetoplastids, this suggests that the Leishmania MVT lysosome and lysosomal microtubule(s) are a reduced cytostome/cytopharynx-like feature.

An ortholog of Plasmodium falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion.

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like organelle, termed the "Vacuolar Compartment/Plant-Like Vacuole" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that contribute to the maturation of invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that the TgCRT protein plays a key role in maintaining the integrity of the parasite's endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in ΔTgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T. gondii.

Leishmania amazonensis hijacks host cell lysosomes involved in plasma membrane repair to induce invasion in fibroblasts.

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.

Phagosome proteomics to study Leishmania's intracellular niche in macrophages.

Intracellular pathogens invade their host cells and replicate within specialized compartments. In turn, the host cell initiates a defensive response trying to kill the invasive agent. As a consequence, intracellular lifestyle implies morphological and physiological changes in both pathogen and host cell. Leishmania spp. are medically important intracellular protozoan parasites that are internalized by professional phagocytes such as macrophages, and reside within the parasitophorous vacuole inhibiting their microbicidal activity. Whereas the proteome of the extracellular promastigote form and the intracellular amastigote form have been extensively studied, the constituents of Leishmania's intracellular niche, an endolysosomal compartment, are not fully deciphered. In this review we discuss protocols to purify such compartments by means of an illustrating example to highlight generally relevant considerations and innovative aspects that allow purification of not only the intracellular parasites but also the phagosomes that harbor them and analyze the latter by gel free proteomics.

Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion.

Successful infection by Trypanosoma cruzi, the agent of Chagas' disease, is critically dependent on host cell invasion by metacyclic trypomastigote (MT) forms. Two main metacyclic stage-specific surface molecules, gp82 and gp90, play determinant roles in target cell invasion in vitro and in oral T. cruzi infection in mice. The structure and properties of gp82, which is highly conserved among T. cruzi strains, are well known. Information on gp90 is still rather sparse. Here, we attempted to fill that gap. gp90, purified from poorly invasive G strain MT and expressing gp90 at high levels, inhibited HeLa cell lysosome spreading and the gp82-mediated internalization of a highly invasive CL strain MT expressing low levels of a diverse gp90 molecule. A recombinant protein containing the conserved C-terminal domain of gp90 exhibited the same properties as the native G strain gp90: it counteracted the host cell lysosome spreading induced by recombinant gp82 and exhibited an inhibitory effect on HeLa cell invasion by CL strain MT. Assays to identify the gp90 sequence associated with the property of downregulating MT invasion, using synthetic peptides spanning the gp90 C-terminal domain, revealed the sequence GVLYTADKEW. These data, plus the findings that lysosome spreading was induced upon HeLa cell interaction with CL strain MT, but not with G strain MT, and that in mixed infection CL strain MT internalization was inhibited by G strain MT, suggest that the inhibition of target cell lysosome spreading is the mechanism by which the gp90 molecule exerts its downregulatory role.

K63-Linked Ubiquitination Targets Toxoplasma gondii for Endo-lysosomal Destruction in IFNγ-Stimulated Human Cells.

Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.

Microfilariae of Brugia malayi Inhibit the mTOR Pathway and Induce Autophagy in Human Dendritic Cells.

Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.

Lysosome biogenesis/scattering increases host cell susceptibility to invasion by Trypanosoma cruzi metacyclic forms and resistance to tissue culture trypomastigotes.

A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is whether the insect-borne and mammalian-stage parasites use similar mechanisms for invasion. To address that question, we analysed the cell invasion capacity of metacyclic trypomastigotes (MT) and tissue culture trypomastigotes (TCT) under diverse conditions. Incubation of parasites for 1 h with HeLa cells in nutrient-deprived medium, a condition that triggered lysosome biogenesis and scattering, increased MT invasion and reduced TCT entry into cells. Sucrose-induced lysosome biogenesis increased HeLa cell susceptibility to MT and resistance to TCT. Treatment of cells with rapamycin, which inhibits mammalian target of rapamycin (mTOR), induced perinuclear lysosome accumulation and reduced MT invasion while augmenting TCT invasion. Metacylic trypomastigotes, but not TCT, induced mTOR dephosphorylation and the nuclear translocation of transcription factor EB (TFEB), a mTOR-associated lysosome biogenesis regulator. Lysosome biogenesis/scattering was stimulated upon HeLa cell interaction with MT but not with TCT. Recently, internalized MT, but not TCT, were surrounded by colocalized lysosome marker LAMP2 and mTOR. The recombinant gp82 protein, the MT-specific surface molecule that mediates invasion, induced mTOR dephosphorylation, nuclear TFEB translocation and lysosome biogenesis/scattering. Taken together, our data clearly indicate that MT invasion is mainly lysosome-dependent, whereas TCT entry is predominantly lysosome-independent.

Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages.

Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.

Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.

Targeted extracellular signal-regulated kinase activation mediated by Leishmania amazonensis requires MP1 scaffold.

Leishmania amazonensis infection promotes alteration of host cellular signaling and intracellular parasite survival, but specific mechanisms are poorly understood. We previously demonstrated that L. amazonensis infection of dendritic cells (DC) activated extracellular signal-regulated kinase (ERK), an MAP-kinase kinase kinase, leading to altered DC maturation and non-healing cutaneous leishmaniasis. Studies using growth factors and cell lines have shown that targeted, robust, intracellular phosphorylation of ERK1/2 from phagolysosomes required recruitment and association with scaffolding proteins, including p14/MP1 and MORG1, on the surface of late endosomes. Based on the intracellular localization of L. amazonensis within a parasitophorous vacuole with late endosome characteristics, we speculated that scaffolding proteins would be important for intracellular parasite-mediated ERK signaling. Our findings demonstrate that MP1, MORG1, and ERK all co-localized on the surface of parasite-containing LAMP2-positive phagolysosomes. Infection of MEK1 mutant fibroblasts unable to bind MP1 demonstrated dramatically reduced ERK1/2 phosphorylation following L. amazonensis infection but not following positive control EGF treatment. This novel mechanism for localization of intracellular L. amazonensis-mediated ERK1/2 phosphorylation required the endosomal scaffold protein MP1 and localized to L. amazonensis parasitophorous vacuoles. Understanding how L. amazonensis parasites hijack host cell scaffold proteins to modulate signaling cascades provides targets for antiprotozoal drug development.

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells.

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca(2+) transients and TGF-ß-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.

IL-17A promotes macrophage effector mechanisms against Trypanosoma cruzi by trapping parasites in the endolysosomal compartment.

The contribution of the IL-23-IL-17A pathway to resistance against extracellular bacterial infections is well established, whereas its role in immunity to intracellular pathogens is much less clear. To analyze the contribution of the IL-23-IL-17A-axis to resistance against Trypanosoma cruzi infection, we infected IL-23p19(-/-) mice and IL-17A(-/-) mice with T. cruzi. Both mouse strains were susceptible to T. cruzi infection despite strong Th1 immune responses. In vitro experiments revealed that IL-17A, but not IL-23, directly stimulates macrophages to internalize T. cruzi parasites by phagocytosis, which is in contrast to the active invasion process normally used by T. cruzi. In contrast to the active entry of parasites into macrophages, the IL-17A-driven phagocytosis prolonged residency of parasites in the endosomal/lysosomal compartment of the macrophage, which subsequently led to eradication of parasites. This IL-17A-dependent mechanism represents a novel function of IL-17A trapping pathogens in endosomal/lysosomal compartments and enhancing exposure time to antimicrobial effectors of the macrophage.

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay.

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.

Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease.

Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling.

Mechanisms of host cell invasion by Trypanosoma cruzi.

One of the more accepted concepts in our understanding of the biology of early Trypanosoma cruzi-host cell interactions is that the mammalian-infective trypomastigote forms of the parasite must transit the host cell lysosomal compartment in order to establish a productive intracellular infection. The acidic environment of the lysosome provides the appropriate conditions for parasite-mediated disruption of the parasitophorous vacuole and release of T. cruzi into the host cell cytosol, where replication of intracellular amastigotes occurs. Recent findings indicate a level of redundancy in the lysosome-targeting process where T. cruzi trypomastigotes exploit different cellular pathways to access host cell lysosomes in non-professional phagocytic cells. In addition, the reversible nature of the host cell penetration process was recently demonstrated when conditions for fusion of the nascent parasite vacuole with the host endosomal-lysosomal system were not met. Thus, the concept of parasite retention as a critical component of the T. cruzi invasion process was introduced. Although it is clear that host cell recognition, attachment and signalling are required to initiate invasion, integration of this knowledge with our understanding of the different routes of parasite entry is largely lacking. In this chapter, we focus on current knowledge of the cellular pathways exploited by T. cruzi trypomastigotes to invade non-professional phagocytic cells and to gain access to the host cell lysosome compartment.