Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life of Host Cell Transcripts by the Parasite.

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

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.

Diseño, síntesis y evaluación biológica de un grupo de pirazinoisoquinolinas y quinolinas con posible actividad esquistosomicida y leishmanicida respectivamente / Design, synthesis and biological evaluation of a group of pyrazinoisoquinolines and quinolines with possible schistosomicidal and leishmanicidal activity respectively

La esquistosomiasis y la leishmaniasis son dos parasitosis con una alta incidencia en el mundo y con la menor cantidad de medicamentos disponibles para sus tratamientos. Para la esquistosomiasis, el praziquantel (PZQ) es la única droga que existe en los actuales momentos contra la enfermedad, mientras que, en el caso de la leishmaniasis, los antimoniales pentavalentes, empleados como drogas de primera línea, son altamente tóxicos o presentan problemas de resistencia. Por ello, esta tesis describe el diseño, la síntesis y los estudios de actividad biológica de un grupo de pirazinoisoquinolinas y quinolinas sustituidas con posible actividad esquistosomicida y leishmanicida respectivamente. Luego de ensayar diversas vías, se logró la síntesis del PZQ y compuestos relacionados (quince compuestos) mediante una secuencia de cinco pasos, con rendimientos entre el 20% y el 60%. El PZQ se obtuvo en un 33%, con un exceso del enantiómero levo, mostrando ser más activo que el PZQ comercial (mezcla racémica). Los compuestos obtenidos, evaluados en cepas de S. mansoni, no mostraron ser más activos que el PZQ, a las dos concentraciones evaluadas. También, se cuantificó la relación entre la estructura química y la actividad biológica (QSAR) de derivados de PZQ reportados en la literatura. En cuanto a los compuestos con posible actividad leishmanicida, también se ensayaron varios métodos de síntesis hasta lograr obtener veintidós quinolínas de los tipos 2-metil, 2-propil, 4-metil-2-propil y 2-alquildiamino con rendimientos entre un 10 y un 70%. Los compuestos evaluados que mostraron una actividad prometedora en promastigotes de L. mexicanafueron la 2-metilquinolina y 6,7-metilendioxi-2-propil-quinolina. En cuanto a los estudios QSAR, no fue posible encontrar una ecuación representativa que relacionara la actividad biológica con la estructura química.

Schistosomiasis and leishmaniasis are two parasitic diseaseswell spread in the world, and at the same time both have very few medications for their treatment. For schistosomiasis, praziquantel (PZQ) is the drug of choice for its treatment, while in the case of leishmaniasis, the pentavalent antimonials used as first line drugs are highly toxic or present resistance problems. This work describes the design, synthesis and biological activity studies of a group of pirazinoisoquinolines and substituted quinolines with a possible schistosomicidal and leishmanicidal activities, respectively. After several intents, it was possible to synthesize PZQ and some related compounds (fifteen) through a sequence of five steps, with yields between 20 and 60%. PZQ was obtained with a yield of 33%, with a levoenantiomeric excess, and showed a better activity than the commercial compound. The related compounds obtained, evaluated against S. mansonistrains did not have an activity comparable to that of PZQ, at the concentrations evaluated. Quantitative structure­activity relationships (QSAR)studieswere also performed with PZQ derivatives reported in the literature.Several ways of synthesis were also probed for the possible leishmanicidal compounds proposed, until it was possible to obtain twenty two quinoline derivatives (of the type 2-methyl-, 2-propyl-, 4-methyl-2-propyl-and 2-alquildiamino-), with yields between10 and 70%. Of the compounds evaluated, two showed promising activity against L. mexicana promastigotes, 2-iimethylquinoline and 6,7-methylendioxi-2-propyl-quinoline. QSAR studies with these compounds did not yield a representative model for the set.

Clonaje de la proteína de choque térmico de 20 kDa de Leishmania amazonensis / Cloning of 20 kDa heat shock protein of Leishmania amazonensis

INTRODUCCIÓN: la inducción de las proteínas de choque térmico constituyen un mecanismo homeostático que protege a las células del efecto destructivo del calor u otras condiciones de estrés ambiental, paralelamente, ellas cumplen importantes funciones celulares. La proteína de choque térmico de 20 kDa se reportó recientemente en Leishmania amazonensis. OBJETIVO: describir la metodología utilizada para realizar el clonaje de las proteínas de choque térmico, lo que permitió acometer estudios de algunas propiedades biológicas. MÉTODOS: la región codificante del gen hsp20 se amplificó mediante la reacción en cadena de la polimerasa con cebadores adecuados. El producto amplificado se clonó inicialmente en el vector pCR2.1 (Invitrogen) y después en el vector de expresión en procariotas pET-28b (Novagen), para obtener proteína recombinante. De manera paralela, el mismo fragmento se clonó en el vector de expresión en eucariotas pcDNA3 (Invitrogen) para obtener un posible preparado vacunal de ADN. Se realizó la secuenciación nucleotídica de los clones obtenidos, con la finalidad de verificar su fidelidad. RESULTADOS: se obtuvieron plásmidos recombinantes que codifican la HSP20 de Leishmania, y permiten la obtención de proteína recombinante y de ADN en forma masiva. CONCLUSIONES: ambos plásmidos fueron útiles para estudiar algunas de las propiedades biológicas de esta proteína. Este acercamiento puede ser de interés en otros trabajos de esta índole y constituir una guía metodológica(AU)

INTRODUCTION: the induction of heat shock proteins is a homeostatic mechanism that protects cells from the deleterious effects of thermal and other environmental stresses. In addition, they have important cell functions. The 20kDa heat shock protein in Leishmania amazonensis was recently reported. OBJECTIVE: to describe the methodology used for cloning of heat shock proteins, which allowed the study of some biological properties. METHODS: the hsp20 gene coding region was amplified by polymerase chain reaction using adequate primers. The amplified product was initially cloned in pCR2.1 vector (Invitrogen) and then in pET-28b vector (Novagen), to obtain recombinant protein. The same fragment was cloned also in the eukariote expression vector pcDNA3 (Invitrogen). The nucleotidic sequencing of the different clones was made, in order to verify their fidelity. RESULTS: the recombinant plasmids that encode HSP20 protein in Leishmania and allow obtaining massively recombinant protein and DNA were produced. CONCLUSIONS: both plasmids were useful to study some of the biological properties of this protein. This approach could be useful for similar research and represent a suitable methodological guideline(AU)

A resposta imuno-inflamatória na leishmaniose tegumentar humana / The immune-inflammatory response in human cutaneous leishmaniasis

Na LTA é reconhecido um espectro de manifestações clínico-munológicas que varia da forma cutânea localizada (LCL) e mucocutânea (LCM), à difusa (LCD). A forma de apresentação clínica é influenciada pela resposta do hospedeiro. A maioria dos pacientes infectados com L. braziliensis desenvolve LCL, uma resposta imuno é montada contra o parasito. Poucos pacientes infectados por L. braziliensis apresentam LCM que é vista como o p!Jlo hiperresponsivo da doença. Raramente, se observa a forma anérgica, LCD, que no Brasil é causada por L. amazonensis. O objetivo deste trabalho foi caracterizar in situ o perfil fenotípico das células do infiltrado inflamatório mononuclear e as citocinas, TNF-a, TGF-B, IFN-y e a enzima iNOS, na tentativa de estabelecer correlações com os váriados aspectos morfológicos da L TA. Foram estudados 43 pacientes, 35 com LCL, 5 com LCM e 3 com LCD. As biópsias de pele e mucosa foram submetidas a estudo histológico e imunohistoquímico pela técnica de imunoperoxidase indireta. As três formas clínicas da L TA mostraram diferenças na composição celular do infiltrado inflamatório. Na LCL os macrófagos foram mais numerosos que T CD4+ e T CD8+, sendo os T CD8+ menos numerosos. Linfócitos T CD8+ foram mais numerosos nas lesões com tempo de evolução maior que 10 semanas ou naquelas que mostraram necrose em granulomas. Na LCM observou se uma equivalência numérica entre estes três fenótipos. Na LCD macrófagos representaram 89% das células do infiltrado inflamatório. Os linfócitos TCD4+ e T CD8+ foram numericamente iguais ou equivalentes. IFN-y foi observado em raras células na LCL e LCM e esteve ausente na LCD. TNF-a foi observado em células mononucleares em torno de vasos, circundando áreas de necrose e apoptose e em células epitelióides de granuloma. TNF-a associado a membrana foi observado em células mononuleares em torno de vasos...

Leishmaniasis mexicana. Consideraciones epidemiológicas, clínicas y terapéuticas para el médico general / Mexican Leishmaniasis. Epidemiological, clinical and therapeutic considerations for the general physician

La Leishmaniasis (L.) es la enfermedad parasitaria producida por el género Leishmania mexicana, transmitida por las hembras de los dípteros conocidos como flebótomos en Europa y Lutzomyia. A partir de 1994, la movilización de tropas en la selva de Chiapas, ha incrementado los riesgos de exposición a la picadura de dichos insectos, por lo que al reincorporarse a sus unidades o instalaciones militares de origen, el personal padece úlceras crónicas cutáneas y, si el médico no tiene en mente este diagnóstico, se retrasa el abordaje clínico y el tratamiento. Por tal motivo, considero de interés realizar la revisión de aspectos epidemiológicos, clínicos y de tratamiento para actualizar la información del médico general

Surface Zn-proteinase as a molecule for defense of Leishmania mexicana amazonensis promastigotes against cytolysis inside macrophage phagolysosomes.

The role of the surface membrane Zn-proteinase in protecting the cellular integrity of the macrophage parasite Leishmania mexicana amazonensis from intraphagolysosomal cytolysis was studied. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth medium. The virulent and attenuated variants of the parasite cells were cloned. Failure of these attenuated parasite cells to survive inside macrophage phagolysosomes is associated with 20- to 50-fold reduction in the expression of surface gp63 protein. In situ inhibition of gp63 proteinase activity inside Leishmania-infected macrophage phagolysosomes with targeted delivery of an inhibitor of gp63 proteinase activity, 1,10-phenanthroline, selectively eliminated intracellular Leishmania amastigotes, further suggesting the importance of this proteinase in phagolysosomal survival of the parasite. An upstream sequence (US) of the gp63 gene was cloned in front of the bacterial chloramphenicol acetyltransferase (CAT) gene in plasmid pCATbasic. Transfection of L. mexicana amazonensis cells with this recombinant plasmid showed that expression of the CAT gene from this US is 15- to 20-fold higher in virulent clones than in avirulent clones of the parasite. Band shift analysis with the cloned US also showed that binding of protein(s) was 15- to 20-fold higher in virulent cell extract than in avirulent cell extract. Coating of attenuated cells or liposomes with proteolytically active gp63 protects them from degradation inside macrophage phagolysosomes. These results suggest a novel mechanism of survival of this phagolysosomal parasite with the help of its surface Zn-proteinase.

Complement resistance of Leishmania amazonensis promastigotes is independent of parasite proteases and lysis of sensitive forms is not due to natural antibodies in normal human serum.

Leishmania amazonensis promastigotes cultivated in vitro differentiate from complement-sensitive to complement-resistant forms. In order to determine the possible involvement of parasite proteases in this process, L. amazonensis promastigotes were collected daily and their proteolytic enzyme patterns analyzed using polacrylamide gels copolymerized with gelatin. Although promastigotes at different growth stages showed differences in protease patterns, these changes did not correlate with their susceptibility to complement. The major protease of promastigotes, gp63, was expressed at the same level throughout culture, regardless of the complement resistance of the promastigotes. Furthermore, inhibitors specific for the classes of proteases found in L. amazonensis promastigotes did not interfere with the complement-mediated killing of promastigotes. We also investigated the binding of natural antibodies to promastigotes. at different stages of growth using ELISA. Although complement-sensitive promastigotes bound significantly more antibodies from fresh normal human serum than complement-resistant promastigotes, equivalent amounts of C3 were detected on their surfaces following complement activation. Moreover, serum depleted of anti-Leishmania antibodies was an efficient in killing promastigotes and the intact serum. These data suggest that the resistance of L. amazonensis to complement killing involves strategies other than that of the regulated expression of endogenous proteases capable of inactivating complement components, or the differential ability to bind natural antibodies that might interfere with complement deposition on the parasite surface.

Complement resistance of Leishmania amazonensis promastigotes is independent of parasite proteases and lysis of sensitive forms is not due to natural antibodies in normal human serum

Leishmania amazonensis promastigotes cultivated in vitro differentiate from complement-sensitive to complement-resistant forms. In order to determine the possible involvement of parasite proteases in this process, L. amazonensis promastigotes were collected daily and their proteolytic enzyme patterns analyzed using polyacrylamide gels copolymerized with gelatin. Although promastigotes at different growth stages showed differences in protease patterns, these changes did not correlate with their susceptibility to complement. The major protease of promastigotes, gp63, was expressed at the same level throughout culture, regardless of the complement resistance of the promastigotes. Furthermore, inhibitors specific for the classes of proteases found in L. amazonensis promastigotes did not interfere with the complement-mediated killing of promastigotes. We also investigated the binding of natural antibodies to promastigotes at different stages of growth using ELISA. Although complement-sensitive promastigotes bound significantly more antibodies from fresh normal human serum than complement-resistant promastigotes, equivalent amounts of C3 were detected on their surfaces following complement activation. Moreover, serum depleted of anti-Leishmania antibodies was as efficient in killing promastigotes as the intact serum. These data suggest that the resistance of L. amazonensis to complement killing involves strategies other than that of the regulated expression endogenous proteases capable of inactivating complement components, or the differential ability to bind natural antibodies that might interfere with complement deposition on the parasite surface.

Uptake of Z-Tyr{125I}-AlaCHN2, an irreversible cysteine proteinase inhibitor, by lesion amastigotes, axenic amastigotes and promastigotes of Leishmania mexicana

Radioiodinated N-benzyloxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[l25I]-AlaCHN2) was previously shown to selectively label two (28 and 31 kDa) Leishmania mexicana cysteine proteinases common to both the promastigote and the amastigote stages. Here we have confirmed the specificity of the compound towards two similar enzymes of axenic L. mexicana amastigotes and demonstrated that lesion amastigotes, axenic amastigotes and stationary promastigotes internalized the l25I-labeled inhibitor at different rates. Uptake of Z-Tyr[l25I]-AlaCHN2 by the parasites, which was not significantly modified by changing the medium pH, was clearly correlated with the binding of the compound to the 28- and 3l-kDa cysteine proteinases, as judged by the specificity of enzyme labeling in gelatin gels and the recovery of 75 per cent or more parasite-associated radioactivity in TCA-insoluble fractions. For all three developmental stages, uptake markedly increased with time and linearly up to 60 min, but throughout the period examined, radiolabel accumulation occurred more efficiently in amastigotes. By 5 h, when values were near or at saturation, radioactivity (in cpm/mug of total protein) associated with lesion amastigotes was 1.8- and 2.9-times that recovered from axenic amastigotes and stationary promastigotes, respectively. Pulse-chase experiments, in which cysteine proteinases were fully blocked with Z-Phe-AlaCHN2 prior to the pulse with Z-Tyr[l25I]-AlaCHN2, showed that labeling of the amastigote enzymes could be partially restored, whereas labeling of promastigote proteinases could not, after a 5-h chase period in inhibitor-free medium.

Efecto del BCG sobre la evolución de la leishmaniasis tegumentaria / Effect of the BCG on the tegumentary leishmaniasis

Durante mucho tiempo, la infección experimental con Leishmania sp. ha sido considerada ser solamente un prototipo para el estudio de las interacciones generales parásito-hospedador; algunos autores han propuesto que la eficiencia terapéutica de la inmunoterapia (BCG más promastigotes de Leishmania mexicana) es igual a la de la quimioterapia (Glucantime). En nuestro modelo experimental la inoculación de BCG en lesiones de leishmaniasis tegumentaria, que teóricamente debería haber provocado una estimulación inmune inespecífica, encontró como hallazgo fundamental, la muerte de todo el lote experimental, que fue inyectado con BCG intralesional, sugiriendose como posible mecanismo el desarrollo de un fenómeno inmunosupresivo, por competencia antigénica

Structural observations on the attachment of promastigotes of Leishmania mexicana amazonensis to the surface of macrophages

Se analiza el proceso de interacción entre el parásito Leishmania mexicana amazonensis y los macrófagos, en experimentos en los cuales se emplea una alta relación parásito/macrófago, mediante el empleo de las microscopías de luz y electrónicas de barrido y transmisión. Los parásitos se adhieren al macrófago pero no son ingeridos por éste. la adherencia se realiza en los promastigotes vivos por el flagelo, pero cuando se emplean amastigotas o promastigotas fijados en glutaraldehido la adherencia se realiza sin orientación específica. la adherencia de los parásitos a la superficie del macrófago induce cambios en la organización estructural de periferia de éste pero no interfiere con la distribución de los sitios de unión de la concanavalina A. La incubación previa de los macrófagos en presencia de citocalasina B o ferritina cationizada no interfiere con la adherencia de los parásitos a la superficie del macrófago

Structural observations on the attachment of promastigotes of Leishmania mexicana amazonensis to the surface of macrophages

Se analiza el proceso de interacción entre el parásito Leishmania mexicana amazonensis y los macrófagos, en experimentos en los cuales se emplea una alta relación parásito/macrófago, mediante el empleo de las microscopías de luz y electrónicas de barrido y transmisión. Los parásitos se adhieren al macrófago pero no son ingeridos por éste. la adherencia se realiza en los promastigotes vivos por el flagelo, pero cuando se emplean amastigotas o promastigotas fijados en glutaraldehido la adherencia se realiza sin orientación específica. la adherencia de los parásitos a la superficie del macrófago induce cambios en la organización estructural de periferia de éste pero no interfiere con la distribución de los sitios de unión de la concanavalina A. La incubación previa de los macrófagos en presencia de citocalasina B o ferritina cationizada no interfiere con la adherencia de los parásitos a la superficie del macrófago (AU)