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Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoan parasite that infects phagocytic and non-phagocytic mammalian cells. At early stages of infection, trypomastigotes, the infective forms of this parasite, localize in a vesicular compartment called the T. cruzi parasitophorous vacuole until the exit of parasites to the host cell cytoplasm where continue their infective cycle. Rab proteins participate in the membrane traffic's molecular machinery, functioning as central regulators of vesicle recognition and transport. In previous work, we demonstrated that endocytic Rabs are key factors of the T. cruzi infection process in non-phagocytic cells, regulating the formation and the maturation of the vacuole. In this work, we identified and characterized other molecular components of the vesicular transport pathways and their participation in the T. cruzi infection. We found that Rab9a and Rab32, two regulators of the endocytic and autophagic pathways, were actively recruited to the T. cruzi vacuoles and favored the late stages of the infective process. The recruitment was specific and dependent on T. cruzi protein synthesis. Interestingly, Rab32 association depends on the presence of Rab9a in the vacuolar membrane, while the inhibition of the cysteine-protease cruzipain, a T. cruzi virulence factor, significantly decreases both Rab9a and Rab32 association with the vacuole. In summary, this work showed for the first time that specific molecules produced and secreted by the parasite can subvert intracellular components of host cells to benefit the infection. These new data shed light on the complex map of interactions between T. cruzi and the host cell and introduce concepts that can be useful in finding new forms of intervention against this parasite in the future.
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Polyclonal B cell activation and the resulting hypergammaglobulinemia are a detrimental consequence of visceral leishmaniasis (VL); however, the mechanisms underlying this excessive production of nonprotective antibodies are still poorly understood. Here, we show that a causative agent of VL, Leishmania donovani, induces CD21-dependent formation of tunneling nanotubule (TNT)-like protrusions in B cells. These intercellular connections are used by the parasite to disseminate among cells and propagate B cell activation, and close contact both among the cells and between B cells and parasites is required to achieve this activation. Direct contact between cells and parasites is also observed in vivo, as L. donovani can be detected in the splenic B cell area as early as 14 days postinfection. Interestingly, Leishmania parasites can also glide from macrophages to B cells via TNT-like protrusions. Taken together, our results suggest that, during in vivo infection, B cells may acquire L. donovani from macrophages via TNT-like protrusions, and these connections are subsequently exploited by the parasite to disseminate among B cells, thus propagating B cell activation and ultimately leading to polyclonal B cell activation. IMPORTANCE Leishmania donovani is a causative agent of visceral leishmaniasis, a potentially lethal disease characterized by strong B cell activation and the subsequent excessive production of nonprotective antibodies, which are known to worsen the disease. How Leishmania activates B cells is still unknown, particularly because this parasite mostly resides inside macrophages and would not have access to B cells during infection. In this study, we describe for the first time how the protozoan parasite Leishmania donovani induces and exploits the formation of protrusions that connect B lymphocytes with each other or with macrophages and glides on these structures from one cell to another. In this way, B cells can acquire Leishmania from macrophages and become activated upon contact with the parasites. This activation will then lead to antibody production. These findings provide an explanation for how the parasite may propagate B cell activation during infection.
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Leishmania donovani , Leishmaniasis Visceral , Humanos , Leishmania donovani/fisiología , Leishmaniasis Visceral/parasitología , MacrófagosRESUMEN
Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.
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Pathogen-specific rewiring of host cell metabolism creates the metabolically adapted microenvironment required for pathogen replication. Here, we investigated the mechanisms governing the modulation of macrophage mitochondrial properties by the vacuolar pathogen Leishmania. We report that induction of oxidative phosphorylation and mitochondrial biogenesis by Leishmania donovani requires the virulence glycolipid lipophosphoglycan, which stimulates the expression of key transcriptional regulators and structural genes associated with the electron transport chain. Leishmania-induced mitochondriogenesis also requires a lipophosphoglycan-independent pathway involving type I interferon (IFN) receptor signaling. The observation that pharmacological induction of mitochondrial biogenesis enables an avirulent lipophosphoglycan-defective L. donovani mutant to survive in macrophages supports the notion that mitochondrial biogenesis contributes to the creation of a metabolically adapted environment propitious to the colonization of host cells by the parasite. This study provides novel insight into the complex mechanism by which Leishmania metacyclic promastigotes alter host cell mitochondrial biogenesis and metabolism during the colonization process. IMPORTANCE To colonize host phagocytes, Leishmania metacyclic promastigotes subvert host defense mechanisms and create a specialized intracellular niche adapted to their replication. This is accomplished through the action of virulence factors, including the surface coat glycoconjugate lipophosphoglycan. In addition, Leishmania induces proliferation of host cell mitochondria as well as metabolic reprogramming of macrophages. These metabolic alterations are crucial to the colonization process of macrophages, as they may provide metabolites required for parasite growth. In this study, we describe a new key role for lipophosphoglycan in the stimulation of oxidative phosphorylation and mitochondrial biogenesis. We also demonstrate that host cell pattern recognition receptors Toll-like receptor 4 (TLR4) and endosomal TLRs mediate these Leishmania-induced alterations of host cell mitochondrial biology, which also require type I IFN signaling. These findings provide new insight into how Leishmania creates a metabolically adapted environment favorable to their replication.
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Leishmania donovani , Biogénesis de Organelos , Macrófagos/metabolismo , GlicoesfingolípidosRESUMEN
Colonization of host phagocytic cells by Leishmania metacyclic promastigotes involves several parasite effectors, including the zinc-dependent metalloprotease GP63. The major mode of action of this virulence factor entails the cleavage/degradation of host cell proteins. Given the potent proteolytic activity of GP63, identification of its substrates requires the adequate preparation of cell lysates to prevent artefactual degradation during cell processing. In the present study, we re-examined the cleavage/degradation of reported GP63 substrates when GP63 activity was efficiently neutralized during the preparation of cell lysates. To this end, we infected bone marrow-derived macrophages with either wild type, Δgp63, and Δgp63+GP63 L. major metacyclic promastigotes for various time points. We prepared cell lysates in the absence or presence of the zinc-metalloprotease inhibitor 1,10-phenanthroline and examined the levels and integrity of ten previously reported host cell GP63 substrates. Inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the cleavage/degradation of several previously described GP63 targets, including PTP-PEST, mTOR, p65RelA, c-Jun, VAMP3, and NLRP3. Conversely, we confirmed that SHP-1, Synaptotagmin XI, VAMP8, and Syntaxin-5 are bona fide GP63 substrates. These results point to the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates. In addition, our results indicate that the role of GP63 in Leishmania pathogenesis must be re-evaluated.
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Leishmania , Proteína Tirosina Fosfatasa no Receptora Tipo 12 , Leishmania/metabolismo , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 12/metabolismo , Proteínas Qa-SNARE/metabolismo , Sinaptotagminas , Serina-Treonina Quinasas TOR/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Factores de Virulencia , Zinc/metabolismoRESUMEN
Macrophages respond to their environment by adopting a predominantly inflammatory or anti-inflammatory profile, depending on the context. The polarization of the subsequent response is regulated by a combination of intrinsic and extrinsic signals and is associated with alterations in macrophage metabolism. Although macrophages are important producers of Wnt ligands, the role of Wnt signaling in regulating metabolic changes associated with macrophage polarization remains unclear. Wnt4 upregulation has been shown to be associated with tissue repair and suppression of age-associated inflammation, which led us to generate Wnt4-deficient bone marrow-derived macrophages to investigate its role in metabolism. We show that loss of Wnt4 led to modified mitochondrial structure, enhanced oxidative phosphorylation, and depleted intracellular lipid reserves, as the cells depended on fatty acid oxidation to fuel their mitochondria. Further we found that enhanced lipolysis was dependent on protein kinase C-mediated activation of lysosomal acid lipase in Wnt4-deficient bone marrow-derived macrophages. Although not irreversible, these metabolic changes promoted parasite survival during infection with Leishmania donovani. In conclusion, our results indicate that enhanced macrophage fatty acid oxidation impairs the control of intracellular pathogens, such as Leishmania. We further suggest that Wnt4 may represent a potential target in atherosclerosis, which is characterized by lipid storage in macrophages leading to them becoming foam cells.
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Aterosclerosis , Fosforilación Oxidativa , Aterosclerosis/metabolismo , Ácidos Grasos/metabolismo , Humanos , Ligandos , Lípidos , Macrófagos/metabolismo , Mitocondrias/metabolismo , Proteína Wnt4/metabolismoRESUMEN
Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite-vector-host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.
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Leishmania infantum , Leishmaniasis Visceral , Parásitos , Animales , Glicoesfingolípidos/metabolismo , Humanos , Leishmaniasis Visceral/metabolismo , Neutrófilos/metabolismo , Parásitos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1ß and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions.
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To colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins to the expansion and functionality of communal vacuoles as well as the replication of the parasite. The differential patterns of recruitment of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor to communal vacuoles harboring Leishmania amazonensis and to individual vacuoles housing L. major led us to further investigate the roles of VAMP3 and VAMP8 in the interaction of Leishmania with its host cell. We show that whereas VAMP8 contributes to the optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither protein has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.
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Leishmania mexicana , Leishmania , Animales , Vivienda , Leishmania/fisiología , Macrófagos/metabolismo , Mamíferos , Vacuolas/parasitología , Proteína 3 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Fragment-based lead discovery has emerged over the last decades as one of the most powerful techniques for identifying starting chemical matter to target specific proteins or nucleic acids in vitro. However, the use of such low-molecular-weight fragment molecules in cell-based phenotypic assays has been historically avoided because of concerns that bioassays would be insufficiently sensitive to detect the limited potency expected for such small molecules and that the high concentrations required would likely implicate undesirable artifacts. Herein, we applied phenotype cell-based screens using a curated fragment library to identify inhibitors against a range of pathogens including Leishmania, Plasmodium falciparum, Neisseria, Mycobacterium, and flaviviruses. This proof-of-concept shows that fragment-based phenotypic lead discovery (FPLD) can serve as a promising complementary approach for tackling infectious diseases and other drug-discovery programs.
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Antiinfecciosos/química , Antiinfecciosos/farmacología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Relación Estructura-ActividadRESUMEN
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) regulate the vesicle transport machinery in phagocytic cells. Within the secretory pathway, Sec22b is an endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-resident SNARE that controls phagosome maturation and function in macrophages and dendritic cells. The secretory pathway controls the release of cytokines and may also impact the secretion of NO, which is synthesized by the Golgi-active inducible NO synthase (iNOS). Whether ERGIC SNARE Sec22b controls NO and cytokine secretion is unknown. Using murine bone marrow-derived dendritic cells, we demonstrated that inducible NO synthase colocalizes with ERGIC/Golgi markers, notably Sec22b and its partner syntaxin 5, in the cytoplasm and at the phagosome. Pharmacological blockade of the secretory pathway hindered NO and cytokine release, and inhibited NF-κB translocation to the nucleus. Importantly, RNA interference-mediated silencing of Sec22b revealed that NO and cytokine production were abrogated at the protein and mRNA levels. This correlated with reduced nuclear translocation of NF-κB. We also found that Sec22b co-occurs with NF-κB in both the cytoplasm and nucleus, pointing to a role for this SNARE in the shuttling of NF-κB. Collectively, our data unveiled a novel function for the ERGIC/Golgi, and its resident SNARE Sec22b, in the production and release of inflammatory mediators.
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Núcleo Celular/metabolismo , Citosol/metabolismo , Células Dendríticas/inmunología , Mediadores de Inflamación/metabolismo , FN-kappa B/metabolismo , Fagosomas/metabolismo , Proteínas R-SNARE/metabolismo , Animales , Células Cultivadas , Citocinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Transporte de Proteínas , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genéticaRESUMEN
Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.
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Interacciones Huésped-Parásitos , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Monocitos/inmunología , Monocitos/parasitología , Fagosomas/inmunología , Fagosomas/parasitología , Glicoesfingolípidos/metabolismo , Interacciones Huésped-Parásitos/inmunología , Leishmania donovani/metabolismo , Leishmania donovani/patogenicidad , Monocitos/metabolismo , NADPH Oxidasas/metabolismo , Virulencia , Factores de VirulenciaRESUMEN
Notch signaling governs crucial aspects of intercellular communication spanning antigen-presenting cells and T-cells. In this study, we investigate how Leishmaniadonovani takes advantage of this pathway to quell host immune responses. We report induction of the Notch ligand Jagged1 in L. donovani-infected bone marrow macrophages (BMMÏs) and subsequent activation of RBPJκ (also known as RBPJ) in T cells, which in turn upregulates the transcription factor GATA3. Activated RBPJκ also associates with the histone acetyltransferase p300 (also known as EP300), which binds with the Bcl2l12 promoter and enhances its expression. Interaction of Bcl2L12 with GATA3 in CD4+ T cells facilitates its binding to the interleukin (IL)-10 and IL-4 promoters, thereby increasing the secretion of these cytokines. Silencing Jagged1 hindered these events in a BMMÏ-T cell co-culture system. Upon further scrutiny, we found that parasite lipophosphoglycan (LPG) induces the host phosphoinositide 3-kinase (PI3K)/Akt pathway, which activates ß-catenin and Egr1, the two transcription factors responsible for driving Jagged1 expression. In vivo morpholino-silencing of Jagged1 suppresses anti-inflammatory cytokine responses and reduces organ parasite burden in L. donovani-infected Balb/c mice, suggesting that L. donovani-induced host Jagged1-Notch signaling skews macrophage-T cell crosstalk into disease-promoting Th2 mode in experimental visceral leishmaniasis.This article has an associated First Person interview with the first author of the paper.
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Leishmania donovani , Leishmaniasis Visceral , Animales , Antiinflamatorios , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-QuinasasRESUMEN
In Leishmania, genetic exchange has been experimentally demonstrated to occur in the sand fly vector and in promastigote axenic cultures through a meiotic-like process. No evidence of genetic exchange in mammalian hosts have been reported so far, possibly due to the fact that the Leishmania species used in previous studies replicate within individual parasitophorous vacuoles. In the present work, we explored the possibility that residing in communal vacuoles may provide conditions favorable for genetic exchange for L. mexicana and L. amazonensis. Using promastigote lines of both species harboring integrated or episomal drug-resistance markers, we assessed whether genetic exchange can occur in axenic cultures, in infected macrophages as well as in infected mice. We obtained evidence of genetic exchange for L. amazonensis in both axenic promastigote cultures and infected macrophages. However, the resulting products of those putative genetic events were unstable as they did not sustain growth in subsequent sub-cultures, precluding further characterization.
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Leishmania mexicana , Leishmania , Leishmaniasis , Parásitos , Animales , Leishmania/genética , Leishmania mexicana/genética , Ratones , Ratones Endogámicos BALB CRESUMEN
The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors in mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild-type (WT), LPG-defective (Δlpg1 mutants), and add-back (Δlpg1 + LPG1) lines to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivoLe. infantum WT parasites attached to the Lu. longipalpis midgut in vitro, with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with the Le. infantum WT or Le. infantum Δlpg1 or Le. infantum Δlpg1 + LPG1 line of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to numbers similar to those seen with the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood-engorged midgut extracts led to mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpisIMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. However, the permissiveness of other sand flies with respect to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector, Lutzomyia longipalpis We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.
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Sistema Digestivo/parasitología , Glicoesfingolípidos/metabolismo , Leishmania infantum/fisiología , Psychodidae/fisiología , Psychodidae/parasitología , Animales , Insectos Vectores/parasitología , Insectos Vectores/fisiología , Leishmania infantum/química , Leishmania infantum/genéticaRESUMEN
On the surface of the Leishmania promastigote, phosphoglycans (PG) such as lipophosphoglycan (LPG), proteophosphoglycan (PPG), free phosphoglycan polymers (PGs), and acid phosphatases (sAP), are dominant and contribute to the invasion and survival of Leishmania within the host cell by modulating macrophage signaling and intracellular trafficking. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by the LPG2 gene. Aiming to investigate the role of PG-containing molecules in Leishmania infantum infection process, herein we describe the generation and characterization of L. infantum LPG2-deficient parasites. This gene was unexpectedly identified as duplicated in the L. infantum genome, which impaired gene targeting using the conventional homologous recombination approach. This limitation was circumvented by the use of CRISPR/Cas9 technology. Knockout parasites were selected by agglutination assays using CA7AE antibodies followed by a lectin (RCA 120). Five clones were isolated and molecularly characterized, all revealing the expected edited genome, as well as the complete absence of LPG and PG-containing molecule expression. Finally, the deletion of LPG2 was found to impair the outcome of infection in human neutrophils, as demonstrated by a pronounced reduction (~83%) in intracellular load compared to wild-type parasite infection. The results obtained herein reinforce the importance of LPG and other PGs as virulence factors in host-parasite interactions.
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Leishmania infantum , Leishmania major , Sistemas CRISPR-Cas , Duplicación de Gen , Edición Génica , Glicoesfingolípidos , Humanos , Leishmania infantum/genética , Proteínas de la Membrana/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Leishmania infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63, and others) that promote survival inside the cells. Extracellular vesicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different Leishmania species to modulate inflammatory responses is scarce, especially from those species causing different clinical manifestations (visceral vs. cutaneous). The purpose of this study was to compare macrophage activation using EVs from three Leishmania species from New World including L. infantum, L. braziliensis, and L. amazonensis. EVs were released from promastigote forms, purified by ultracentrifugation and quantitated by Nanoparticle Tracking Analysis (NTA) prior to murine macrophage exposure. NTA analysis did not show any differences in the EV sizes among the strains. EVs from L. braziliensis and L. infantum failed to induce a pro-inflammatory response. EVs from both L. infantum WT and LPG-deficient mutant (LPG-KO) did not show any differences in their interaction with macrophages, suggesting that LPG solely was not determinant for activation. On the other hand, EVs from L. amazonensis were immunomodulatory inducing NO, TNF-α, IL-6, and IL-10 via TLR4 and TLR2. To determine whether such activation was related to NF-κB p65 translocation, THP-1 macrophage cells were exposed to EVs. In the same way, only EVs from L. amazonensis exhibited a highly percentage of cells positive for NF-κB. Our results suggest an important role of EVs in determining the pattern of immune response depending on the parasite species. For L. infantum, LPG was not determinant for the activation.