RESUMEN

Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.

Asunto(s)

Catalasa/genética , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/patogenicidad , Estadios del Ciclo de Vida/genética , Proteínas Protozoarias/genética , Factores de Virulencia/genética , Animales , Catalasa/metabolismo , Células Cultivadas , Femenino , Leishmania mexicana/genética , Ratones , Ratones Endogámicos BALB C , Psychodidae/parasitología , Teschovirus/genética , Virulencia , Factores de Virulencia/metabolismo

RESUMEN

Leishmaniasis has been considered as emerging and re-emerging disease, and its increasing global incidence has raised concerns. The great clinical diversity of the disease is mainly determined by the species. In several American countries, tegumentary leishmaniasis (TL) is associated with both Leishmania amazonensis and L. braziliensis, while visceral leishmaniasis (VL) is associated with L. (L.) infantum. The major molecules that determine the most diverse biological variations are proteins. In the present study, through a DIGE approach, we identified differentially abundant proteins among the species mentioned above. We observed a variety of proteins with differential abundance among the studied species; and the biological networks predicted for each species showed that many of these proteins interacted with each other. The prominent proteins included the heat shock proteins (HSPs) and the protein network involved in oxide reduction process in L. amazonensis, the protein network of ribosomes in L. braziliensis, and the proteins involved in energy metabolism in L. infantum. The important proteins, as revealed by the PPI network results, enrichment categories, and exclusive proteins analysis, were arginase, HSPs, and trypanothione reductase in L. amazonensis; enolase, peroxidoxin, and tryparedoxin1 in L. braziliensis; and succinyl-CoA ligase [GDP -forming] beta-chain and transaldolase in L. infantum.

Asunto(s)

Leishmania braziliensis/patogenicidad , Leishmania infantum/patogenicidad , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/parasitología , Proteínas Protozoarias/metabolismo , Biología Computacional , Humanos , Leishmania braziliensis/metabolismo , Leishmania infantum/metabolismo , Leishmania mexicana/metabolismo , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteínas Protozoarias/aislamiento & purificación

RESUMEN

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1flox/Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1C134A, was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania.

Asunto(s)

Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/fisiología , Leishmania mexicana/enzimología , Leishmaniasis Cutánea/parasitología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Eliminación de Gen , Genes Esenciales , Humanos , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Virulencia/genética

RESUMEN

New anti-infective agents are urgently needed to fight microbial resistance. Methicillin-resistant Staphylococcus aureus (MRSA) strains are particularly responsible for complicated pathologies that are difficult to treat due to their virulence and the formation of persistent biofilms forming a complex protecting shell. Parasitic infections caused by Trypanosoma brucei and Leishmania mexicana are also of global concern, because of the mortality due to the low number of safe and effective treatments. Female inflorescences of hop produce specialized metabolites known for their antimicrobial effects but underexploited to fight against drug-resistant microorganisms. In this study, we assessed the antimicrobial potential of phenolic compounds against MRSA clinical isolates, T. brucei and L. mexicana. By fractionation process, we purified the major prenylated chalcones and acylphloroglucinols, which were quantified by UHPLC-UV in different plant parts, showing their higher content in the active flowers extract. Their potent antibacterial action (MIC < 1 µg/mL for the most active compound) was demonstrated against MRSA strains, through kill curves, post-antibiotic effects, anti-biofilm assays and synergy studies with antibiotics. An antiparasitic activity was also shown for some purified compounds, particularly on T. brucei (IC50 < 1 to 11 µg/mL). Their cytotoxic activity was assessed both on cancer and non-cancer human cell lines.

Asunto(s)

Antiinfecciosos/química , Productos Biológicos/química , Humulus/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Antiinfecciosos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Biopelículas/efectos de los fármacos , Productos Biológicos/farmacología , Humanos , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/patogenicidad , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Pruebas de Sensibilidad Microbiana , Enfermedades Parasitarias/tratamiento farmacológico , Enfermedades Parasitarias/parasitología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/patogenicidad

RESUMEN

Cutaneous leishmaniasis is a neglected tropical disease characterized by disfiguring skin lesions. Current chemotherapeutic options depend on toxic, expensive drugs that are both difficult to administer and becoming less effective due to increasing levels of resistance. In comparison, thermotherapy displays greater patient compliance and less adverse systemic effects, but there are still significant issues associated with this. The procedure is painful, requiring local anaesthetic, and is less effective against large lesions. Using nanoparticles to controllably generate heat in a localized manner may provide an alternative solution. Here we evaluate magnetic hyperthermia, using iron oxide magnetic nanoparticles, as a localized, heat-based method to kill the human-infective parasite in vitro. We assessed the effectiveness of this method against the differentiated, amastigote form of the parasite using three distinct viability assays: PrestoBlue, Live/Dead stain and a novel luciferase-based assay. Changes in amastigote morphology and ultrastructure were assessed by immunofluorescence, scanning and transmission electron microscopy. Our findings show that magnetic hyperthermia is an effective method to kill host-infective amastigotes, with morphological changes consistent with heat treatment. This method has the potential to be a step-change for research into new therapeutic options that moves away from the expensive chemotherapeutics currently dominating the research climate.

Asunto(s)

Hipertermia Inducida/métodos , Leishmania mexicana/patogenicidad , Nanopartículas de Magnetita/química , Nanopartículas/química , Supervivencia Celular/fisiología , Citometría de Flujo , Humanos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente

RESUMEN

BACKGROUND: Leishmaniasis is a neglected disease endemic in tropical and subtropical areas, with an incidence about 1.6 million cases/year. The first-line treatment of this disease is pentavalent antimony, and the second-line are pentamidine and amphotericin B. All the treatments available cause severe side effects and often have difficulty in accessing parasites within infected cells. STUDY QUESTION: This study aimed to determine if the use of nanoparticles loaded with meglumine antimoniate could reach and targeting infected organs with leishmaniasis, reducing the dosage used and promoting less adverse effects. STUDY DESIGN: This study was performed comparing the meglumine nanoparticle in two experimental groups. The first one healthy mice and the second one inducted mice (leishmaniasis). MEASURES AND OUTCOMES: The nanoparticles loaded with meglumine antimoniate (nanoantimony) were prepared by double-emulsion solvent evaporation method and showed a size of about 150-200 nm. BALB/c mice infected or not with Leishmania amazonensis (cutaneous leishmaniasis model) or Leishmania infantum (visceral leishmaniasis model) was used to access the biodistribution of nanoantimony and meglumine antimoniate labeled with technetium-99m. RESULTS: The biodistribution profiles showed a preferential targeting of the nanoparticles to the liver, spleen, and lungs. Because these are the main organs infected, the nanoparticle may be used for this purpose. The results for cutaneous leishmaniasis showed a low uptake by the lesion (infected region). CONCLUSIONS: The results demonstrated the potential use of these nanoparticles to improve the efficacy of meglumine antimoniate in the treatment of visceral leishmaniasis, indicating their potential as an alternative therapeutic strategy for leishmaniasis infections.

Asunto(s)

Antiprotozoarios/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológico , Antimoniato de Meglumina/administración & dosificación , Animales , Modelos Animales de Enfermedad , Humanos , Leishmania infantum/patogenicidad , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/parasitología , Leishmaniasis Visceral/parasitología , Antimoniato de Meglumina/farmacocinética , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Poliésteres/química , Tecnecio/química , Distribución Tisular , Resultado del Tratamiento

RESUMEN

Glucose transporters are important for viability and infectivity of the disease-causing amastigote stages of Leishmania mexicana The Δgt1-3 null mutant, in which the 3 clustered glucose transporter genes, GT1, GT2, and GT3, have been deleted, is strongly impaired in growth inside macrophages in vitro We have now demonstrated that this null mutant is also impaired in virulence in the BALB/c murine model of infection and forms lesions considerably more slowly than wild-type parasites. Previously, we established that amplification of the PIFTC3 gene, which encodes an intraflagellar transport protein, both facilitated and accompanied the isolation of the original Δgt1-3 null mutant generated in extracellular insect-stage promastigotes. We have now isolated Δgt1-3 null mutants without coamplification of PIFTC3 These amplicon-negative null mutants are further impaired in growth as promastigotes, compared to the previously described null mutants containing the PIFTC3 amplification. In contrast, the GT3 glucose transporter plays an especially important role in promoting amastigote viability. A line that expresses only the single glucose transporter GT3 grows as well inside macrophages and induces lesions in animals as robustly as do wild-type amastigotes, but lines expressing only the GT1 or GT2 transporters replicate poorly in macrophages. Strikingly, GT3 is restricted largely to the endoplasmic reticulum in intracellular amastigotes. This observation raises the possibility that GT3 may play an important role as an intracellular glucose transporter in the infectious stage of the parasite life cycle.IMPORTANCE Glucose transport plays important roles for in vitro growth of insect-stage promastigotes and especially for viability of intramacrophage mammalian host-stage amastigotes of Leishmania mexicana However, the roles of the three distinct glucose transporters, GT1, GT2, and GT3, in parasite viability inside macrophages and virulence in mice have not been fully explored. Parasite lines expressing GT1 or GT2 alone were strongly impaired in growth inside macrophages, but lines expressing GT3 alone infected macrophages and caused lesions in mice as robustly as wild-type parasites. Notably, GT3 localizes to the endoplasmic reticulum of intracellular amastigotes, suggesting a potential role for salvage of glucose from that organelle for viability of infectious amastigotes. This study establishes the unique role of GT3 for parasite survival inside host macrophages and for robust virulence in infected animals.

Asunto(s)

Retículo Endoplásmico/parasitología , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Leishmania mexicana/patogenicidad , Proteínas Protozoarias/genética , Animales , Línea Celular , Femenino , Técnicas de Inactivación de Genes , Leishmania mexicana/genética , Estadios del Ciclo de Vida , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Mutación , Virulencia

RESUMEN

Lutzomyia longipalpis is the main vector of visceral leishmaniasis (VL) in America. Physiological and molecular mechanisms of Leishmania infection in sand flies have been studied during the first gonotrophic cycle. There are few studies about these interactions during the second gonotrophic cycle mainly because of the difficulties maintaining sand flies through sequential feeds. Here we standardized conditions to perform the second blood feed efficiently, and our results show that oviposition is an essential factor for the success of multiple feeds. We evaluated the impact of the second blood meal on longevity, protein digestion, trypsin activity, and Leishmania mexicana development within L. longipalpis gut. Mortality of blood-fed females increases after second blood meal as compared to sugar-fed females. Trypsin activity was lower during the second gonotrophic cycle. However, no difference in protein intake was observed between blood meals. There was no difference in the population size of Leishmania in the gut after both blood meals. In this work, we presented an optimized protocol for obtaining sufficient numbers of sand fly females fed on a second blood meal, and we described some physiological and parasitological aspects of the second gonotrophic cycle which might influence the vectorial competence of sand flies.

Asunto(s)

Leishmaniasis/parasitología , Longevidad/fisiología , Oviposición/fisiología , Psychodidae/patogenicidad , Animales , Vectores de Enfermedades , Femenino , Tracto Gastrointestinal/parasitología , Insectos Vectores/fisiología , Leishmania mexicana/patogenicidad , Leishmaniasis/transmisión , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/transmisión , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/transmisión

RESUMEN

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

Asunto(s)

Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/metabolismo , Leishmaniasis Cutánea/parasitología , Metaboloma , Transcetolasa/metabolismo , Virulencia , Animales , Glucólisis , Estadios del Ciclo de Vida , Metabolómica , Ratones , Ratones Endogámicos BALB C , Monocitos/metabolismo , Monocitos/parasitología , Estrés Oxidativo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Eliminación de Secuencia , Transcetolasa/genética

RESUMEN

Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources.

Asunto(s)

Aminoácidos/metabolismo , Leishmania mexicana/metabolismo , Leishmaniasis Cutánea/parasitología , Macrófagos/parasitología , Animales , Carbono/metabolismo , Ciclo del Ácido Cítrico , Modelos Animales de Enfermedad , Femenino , Glucosa/metabolismo , Humanos , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Virulencia

RESUMEN

Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.

Asunto(s)

Sistemas CRISPR-Cas , Genes Protozoarios , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Animales , Simulación por Computador , Femenino , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes/métodos , Humanos , Insectos Vectores/parasitología , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos BALB C , Psychodidae/parasitología , Virulencia/genética

RESUMEN

Cutaneous leishmaniasis is a neglected tropical disease, causing a spectrum of clinical manifestations varying from self-healing to unhealing lesions that may be very difficult to treat. Emerging evidence points to a detrimental role for neutrophils during the first hours following infection with many distinct Leishmania species (spp.) at a time when the parasite is in its nonreplicative promastigote form. Neutrophils have also been detected at later stages of infection in unhealing chronic cutaneous lesions. However, the interactions between these cells and the replicative intracellular amastigote form of the parasite have been poorly studied. Here, we show that Leishmaniamexicana amastigotes are efficiently internalized by neutrophils and that this process has only a low impact on neutrophil activation and apoptosis. In neutrophils, the amastigotes were found in acidified vesicles. Furthermore, within cutaneous unhealing lesions, heavily infected neutrophils were found with up to 6 parasites per cell. To investigate if the amastigotes could replicate within neutrophils, we generated photoconvertible fluorescent parasites. With the use of flow cytometry imaging and time-lapse microscopy, we could demonstrate that a subset of parasites replicated within neutrophils. Overall, our data reveal a novel role for neutrophils that can act as a niche for parasite replication during the chronic phase of infection, thereby contributing to disease pathology.

Asunto(s)

División Celular , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Cutánea/parasitología , Estadios del Ciclo de Vida/genética , Neutrófilos/parasitología , Organismos Modificados Genéticamente/crecimiento & desarrollo , Animales , Femenino , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Interacciones Huésped-Parásitos/inmunología , Leishmania mexicana/patogenicidad , Leishmania mexicana/ultraestructura , Leishmaniasis Cutánea/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/ultraestructura , Fagocitosis , Procesos Fotoquímicos , Imagen de Lapso de Tiempo

RESUMEN

BACKGROUND: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. METHODOLOGY/PRINCIPAL FINDINGS: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. CONCLUSIONS/SIGNIFICANCE: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Leishmania mexicana/patogenicidad , Macrófagos/parasitología , Proteínas Protozoarias/metabolismo , Psychodidae/parasitología , Animales , Femenino , Proteínas de Unión al GTP/genética , Regulación del Desarrollo de la Expresión Génica , Insectos Vectores/parasitología , Leishmania mexicana/genética , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Virulencia

RESUMEN

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Asunto(s)

Leishmania mexicana/genética , Leishmaniasis Cutánea/parasitología , Animales , Modelos Animales de Enfermedad , Femenino , Leishmania mexicana/patogenicidad , Ratones , Ratones Endogámicos BALB C , Fenotipo , Factores de Tiempo

RESUMEN

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Asunto(s)

Animales , Femenino , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/parasitología , Modelos Animales de Enfermedad , Fenotipo , Factores de Tiempo , Ratones Endogámicos BALB C

RESUMEN

A Leishmaniose Cutânea Difusa (LCD) é uma manifestação clínica. rara causada pela Leishmania amazonensis que é caracterizada por uma resposta celular. parasitária ineficiente e macrófagos intensamente parasitados nas lesões cutâneas.. Mediadores lipídicos e seus precursores desempenham um papel crucial durante a. infecção por Leishmania. Estudos prévios demonstram que pacientes com leishmaniose. tegumentar, exibem um distinto balanço de eicosanoides in situ e sistêmico.. Recentemente, demonstrou-se que mediadores lipídicos especializados na pró-resolução. desempenham um papel crítico na redução de processos inflamatórios patológicos. induzindo a restauração da homeostasia em diferentes modelos experimentais. Entre. esses mediadores, as resolvinas da série D exibem potente atividade anti-inflamatória e. imuno-regulatória que inclui a inibição da quimiotaxia leucocitária e bloqueio na. produção de citocinas pró-inflamatórias. No entanto, ainda é desconhecido se as. resolvinas desempenham um papel significativo no estabelecimento e persistência da. infecção por Leishmania. OBJETIVO: Nesse estudo, avaliamos os níveis circulantes. de Resolvina D1 (RvD1) em pacientes com leishmaniose tegumentar apresentando a. forma clínica cutânea localizada (LCL) ou difusa. RESULTADOS: Nossos resultados. demonstram que pacientes com LCD apresentam maiores níveis plasmáticos de RvD1. quando comparados a LCL ou controles endêmicos. Além disso, os níveis séricos de. RvD1 em pacientes com LCD se correlacionam positivamente com a Arginase I e TGF-. β, enquanto que inversamente com os níveis sistêmicos de TNF-α. Experimentos. adicionais in vitro utilizando macrófagos humanos revelaram que a RvD1 promove a. replicação intracelular da L. amazonensis por um mecanismo associado a indução da. enzima heme oxigenase-1. CONCLUSÃO: Os resultados sugerem que a via de. produção da RvD1 pode servir como uma potencial estratégia terapêutica para os. pacientes com LCD.

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INTRODUCTION: Diffuse Cutaneous Leishmaniasis (DCL) is a rare clinical manifestation caused by Leishmania amazonensis that is characterized by an inefficient parasite-specific cellular responses and heavily parasitized macrophages in skin lesions. Lipid mediators and their precursors play a crucial role during Leishmania infection. Previous works have shown that patients with cutaneous leishmaniasis exhibit a distinct in situ and systemic balance of this eicosanoids. Recently, pro-resolution lipid mediators have been shown to play critical role in dampening pathological inflammatory processes to reestablish homeostasis in a diverse range of experimental settings. Among these mediators, resolvins from D series have been described to exhibit potent antiinflammatory and immune-regulatory activities that include inhibition of leukocyte chemotaxis and blockage on the production of proinflammatory cytokines. However, whether resolvins play significant roles in establishment and persistence of Leishmania infection is currently unknown. AIM: We addressed this question by assessing circulating levels of resolvin D1 (RvD1) in tegumentary leishmaniasis patients presenting localized cutaneous leishmaniasis (LCL) or diffuse disease. RESULTS: We found that DCL patients have higher plasma levels of RvD1 when compared with LCL patients or endemic controls. In addition, the levels of this mediator were positively correlated with arginase-I and TGF-β and were negatively correlated with TNF-α levels. Additional in vitro experiments using primary human macrophages revealed that resolvin D1 promotes the intracellular L. amazonensis replication for a mechanism dependent on induction of heme oxygenase-1 enzyme. CONCLUSION: These results indicate that targeting RvD1 could serve as potential strategy for DCL patients.

Asunto(s)

Humanos , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea Difusa/diagnóstico , Leishmaniasis Cutánea Difusa/inmunología , Leishmaniasis Cutánea Difusa/parasitología , Leishmaniasis Cutánea Difusa/patología , Leishmaniasis Cutánea Difusa/prevención & control , Leishmaniasis Cutánea Difusa/sangre , Leishmaniasis Cutánea Difusa/transmisión

RESUMEN

BACKGROUND: Coordination compounds of pentavalent antimony have been, and remain, the first-line drugs in leishmaniasis treatment for >70 years. Molecular forms of Sb (V) complexes are commercialized as sodium stibogluconate (Pentostam®) and meglumine antimoniate (MA) (Glucantime®). Ever-increasing drug resistance in the parasites limits the use of antimonials, due to the low drug concentrations being administered against high parasitic counts. Sb5+ toxicity provokes severe side effects during treatment. To enhance therapeutic potency and to increase Sb (V) concentration within the target cells, we decided to try a new active substance form, a hydrosol of Sb2O5·nH2O nanoparticles (NPs), instead of molecular drugs. METHODOLOGY/PRINCIPAL FINDINGS: Sb2O5·nH2O NPs were synthesized by controlled SbCl5 hydrolysis in a great excess of water. Sb2O5·nH2O phase formation was confirmed by X-ray diffraction. The surface of Sb (V) NPs was treated with ligands with a high affinity for target cell membrane receptors. The mean particle size determined by dynamic light scattering and transmission electron microscopy was ~35-45 nm. In vitro tests demonstrated a 2.5-3 times higher antiparasitic activity of Sb (V) nanohybrid hydrosols, when compared to MA solution. A similar comparison for in vivo treatment of experimental cutaneous leishmaniasis with Sb5+ nanohybrids showed a 1.75-1.85 times more effective decrease in the lesions. Microimages of tissue fragments confirmed the presence of NPs inside the cytoplasm of infected macrophages. CONCLUSION/SIGNIFICANCE: Sb2O5·nH2O hydrosols are proposed as a new form of treatment for cutaneous leishmaniasis caused by Leishmania amazonensis. The NPs penetrate directly into the affected cells, creating a high local concentration of the drug, a precondition to overcoming the parasite resistance to molecular forms of pentavalent antimonials. The nanohybrids are more effective at a lower dose, when compared to MA, the molecular drug. Our data suggest that the new form of treatment has the potential to reduce and simplify the course of cutaneous leishmaniasis treatment. At the same time, Sb2O5·nH2O hydrosols provide an opportunity to avoid toxic antimony (V) spreading throughout the body.

Asunto(s)

Antimonio/farmacología , Antiprotozoarios/química , Antiprotozoarios/farmacología , Nanopartículas/química , Óxidos/farmacología , Animales , Antimonio/química , Evaluación Preclínica de Medicamentos/métodos , Dispersión Dinámica de Luz , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/tratamiento farmacológico , Masculino , Mesocricetus , Microscopía Electrónica de Transmisión , Óxidos/química , Tamaño de la Partícula , Difracción de Rayos X

RESUMEN

Neurological symptoms have been associated with Leishmania infection, however little is known about how the nervous system is affected in leishmaniasis. This work aimed to analyze parasitic load, production of cytokines/neurotrophins in the prefrontal cortex and behavioral changes in BALB/c mice infected with Leishmania amazonensis. At 2 and 4months post-infection, infected mice showed a decrease in IFN-γ, IL-1, IL-6, IL-4, IL-10 cytokines and BDNF and NGF neurotrophins in prefrontal cortex associated with increased anxiety behavior. Parasite DNA was found in brain of all animals at 4months post-infection, when the levels of IBA-1 (activated macrophage/microglia marker) and TNF-α was increased in the prefrontal cortex. However TNF-α returned to normal levels at 6months post-infection suggesting a neuroprotective mechanism.

Asunto(s)

Corteza Cerebral/metabolismo , Citocinas/metabolismo , Leishmaniasis/complicaciones , Leishmaniasis/patología , Trastornos Mentales/etiología , Factores de Crecimiento Nervioso/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Corteza Cerebral/parasitología , ADN Protozoario/genética , ADN Protozoario/metabolismo , Modelos Animales de Enfermedad , Conducta Exploratoria , Regulación de la Expresión Génica , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Leishmaniasis/microbiología , Masculino , Aprendizaje por Laberinto/fisiología , Trastornos Mentales/parasitología , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Piel/patología , Factores de Tiempo

RESUMEN

Cysteine peptidases play a central role in the biology of Leishmania. In this work, we sought to further elucidate the mechanism(s) by which the cysteine peptidase CPB contributes to L. mexicana virulence and whether CPB participates in the formation of large communal parasitophorous vacuoles induced by these parasites. We initially examined the impact of L. mexicana infection on the trafficking of VAMP3 and VAMP8, two endocytic SNARE proteins associated with phagolysosome biogenesis and function. Using a CPB-deficient mutant, we found that both VAMP3 and VAMP8 were down-modulated in a CPB-dependent manner. We also discovered that expression of the virulence-associated GPI-anchored metalloprotease GP63 was inhibited in the absence of CPB. Expression of GP63 in the CPB-deficient mutant was sufficient to down-modulate VAMP3 and VAMP8. Similarly, episomal expression of GP63 enabled the CPB-deficient mutant to establish infection in macrophages, induce the formation of large communal parasitophorous vacuoles, and cause lesions in mice. These findings implicate CPB in the regulation of GP63 expression and provide evidence that both GP63 and CPB are key virulence factors in L. mexicana.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/metabolismo , Metaloendopeptidasas/biosíntesis , Proteínas Protozoarias/metabolismo , Animales , Western Blotting , Cisteína/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Péptido Hidrolasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virulencia , Factores de Virulencia/metabolismo

RESUMEN

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Asunto(s)

Proteínas del Citoesqueleto/genética , Flagelos/genética , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Leishmaniasis/genética , Macrófagos/parasitología , Proteínas Protozoarias/genética , Animales , Citocinesis/genética , Flagelos/parasitología , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Leishmaniasis/parasitología , Leishmaniasis/patología , Ratones , Mutación