Riboflavin Transporter in Trypanosomatids: Novel Therapeutic Target

La riboflavina (vitamina B2) es un nutriente esencial para todo tipo celular estudiado, siendo elprecursor de los cofactores flavin mononucleótido (FMN) y flavin adenin dinucleótido (FAD). Losmetazoos, incluyendo al humano, carecen de la vía biosintética de riboflavina y la obtienen de ladieta a través de transportadores específicos (familia SLC52). Muchos patógenos sintetizanriboflavina de novo mientras otros la incorporan a través de transportadores específicos,estructuralmente diferentes de los transportadores presentes en los metazoos. Por laesencialidad de la riboflavina y las diferencias entre patógenos y hospedadores, las vías deobtención de riboflavina fueron propuestas como potenciales blancos terapéuticos contra lospatógenos. Los tripanosomátidos son un grupo monofilético comprendiendo parásitos responsables degraves enfermedades en humanos tales como Trypanosoma cruzi (Enfermedad de Chagas), Trypanosoma brucei (Tripanosomiasis Humana Africana) y Leishmania spp. (Leishmaniasiscutánea, mucocutánea y visceral). Otros tripanosomátidos son fitoparásitos, como Phytomonasspp., generando grandes pérdidas en economías regionales de Sudamérica. En esta tesis se estudió metabolismo (posibles rutas de obtención por biosíntesis y/o transporte)de riboflavina en tripanosomátidos así como su rol biológico en los distintos estadíos celulares. Nuestros resultados mostraron que las flavinas son esenciales para la proliferación detripanosomátidos. La riboflavina es incorporada a través de un mecanismo mediado por un/ostransportador/es saturable/s. Más aún, procesos biológicos de T. cruzi como la metaciclogénesisy replicación de epimastigotes y de amastigotes en células de mamíferos son dependientes deltransporte de riboflavina. Mediante análisis bioinformáticos, identificamos una nueva familia detransportadores de riboflavina a la que llamamos “RibJ” y caracterizamos funcionalmente lostransportadores RibJ de T. cruzi y T. brucei. Interesantemente, los análisis filogenéticos mostraronque RibJ es específica para tripanosomátidos y no está relacionada con ninguna de las 3 familiasdescriptas en eucariotas. Dado que riboflavina es esencial para tripanosomátidos, que se obtienepor transporte y que RibJ es estructuralmente distinto a los transportadores de otros eucariotas, incluidos los hospedadores, RibJ puede postularse como nuevo blanco para el diseño de drogascon potencial uso en el desarrollo de terapias tripanocidas.Riboflavin (vitamin B2) is an essential nutrient for all studied living cells, being the precursor offlavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. Metazoa, includinghumans, lack the riboflavin biosynthetic pathway and obtain this vitamin from the diet throughits riboflavin transporter (SLC52 family). Many pathogens synthesize riboflavin de novo whileothers incorporate it through specific riboflavin transporters, structurally different from thosepresent in metazoans. For these reasons, the riboflavin pathways have been proposed astherapeutic targets against pathogens. Trypanosomatids are a monophyletic group encompassing parasites responsible of serioushuman diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Human African Trypanosomiasis) and Leishmania spp. (cutaneous, mucocutaneous, and visceralleishmaniasis). Other ones are phytoparasites, as Phytomonasspp., generating regional economiclosses in South America. In this Thesis, we have studied the metabolism (biosynthesis and transport) of riboflavin intrypanosomatids, defining the biological role of this vitamin at the different cellular stages. Ourresults show that flavins are essential for trypanosomatids proliferation, and that riboflavin isincorporated through a saturable carrier-mediated process. Moreover, we have found thatparasites proliferation and T. cruzi metacyclogenesis and amastigote replication in mammaliancells are dependent on flavin transport. By bioinformatics analysis, we have identified a novelfamily of riboflavin transporters, which we named RibJ, functionally characterizing two relevantmembers from this family: TcRibJ and TbRibJ from T. cruzi and T. brucei, respectively. Interestingly, the phylogenetic analysis show that RibJ is specific for trypanosomatids and it is notrelated with the other three families described in eukaryotic cells. This work highlights theessentiality of riboflavin metabolism for trypanosomatids and presents RibJ as an attractive targetfor drug design. Specifically blockage of riboflavin transport may lead to novel therapeuticsagainst trypanosomiasis.Fil: Balcazar, Darío Emmanuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Morphological and molecular characterization of Geraldius galapagoensis (Nematoda: Chambersiellidae) associated with lichens in Argentina

Lichens are symbiotic organisms, usually composed of a fungal partner, the mycobiont, and one or more photosynthetic partners, the photobiont, which is most often either a green alga or a cyanobacterium, that harbor a diverse community of invertebrates such as rotifers, tardigrades, mites, springtails, crustaceans, and nematodes. In this work, we isolated the nematode Geraldius galapagoensis (Chambersiellidae) associated with the lichen Hyperphyscia syncolla (Physciaceae) in a region of Buenos Aires Province, Argentina. This species was discovered in a tropical forest of Ecuador and is characterized mainly by a head offset by a constriction from the rest of the body, a esophagus with a cylindrical pharyngeal corpus without a median bulb, a narrow isthmus and an oval basal pharyngeal bulb with strong transverse/butterfly valve apparatus, a tail curved ventrally, ending in dorsally hooked end; the male with seven pairs of latero-ventral pre-anal papillae and three pairs of post-anal in the following positions: one pair latero-ventral and two pairs dorso-lateral and two slightly curved spicules with asymmetric manubrium with an anterior extension. The comparison of the morphometrics of our G. galapagoensis male with that of the original description shows that the body length is shorter, as are the distance of the excretory pore to the anterior end and the tail. On the other hand, the distance from the anterior end to the nerve ring and the esophagus length are greater. The head width, body diameter and spicule length are quite similar. We provide a morphological and morphometrical characterization of a G. galapagoensis second isolate and the first world report of molecular sequences belonging to this species

Identification of levothyroxine antichagasic activity through computer-aided drug repurposing

Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.Facultad de Ciencias Exacta

Detection of Rickettsia spp. in ectoparasites of cricetid rodents from Gran La Plata, Buenos Aires Province, Argentina

Rickettsia spp. are obligate intracellular Gram-negative bacteria, worldwide distributed. The genus includes more than 20 species, many of them causing a group of diseases in humans and animals known as Rickettsiosis, usually transmitted by arthropod vectors. Although in Argentina Rickettsiosis have a low prevalence, clinical cases have been reported in Gran La Plata area. Rodents are usual hosts of ectoparasites, some of which have been involved in the enzootic life cycle of rickettsial species. The aim of this work was to detect bacteria of the genus Rickettsia in fleas, mites and ticks associated with cricetids in Gran La Plata.Para acceder a la videoconferencia completa, hacer clic en "Enlace externo".Sociedad Latinoamericana de Ecología de Vectore

Identification of levothyroxine antichagasic activity through computer-aided drug repurposing

Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.Facultad de Ciencias Exacta

Smoking flies: testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster

Studies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of ‘smoking flies’. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.Centro de Investigaciones CardiovascularesCentro de Estudios Parasitológicos y de Vectore

Broadening the spectrum of ivermectin: Its effect on Trypanosoma cruzi and related trypanosomatids

Chagas disease is an endemic American parasitosis, caused by Trypanosoma cruzi. The current therapies, benznidazole (BZN) and nifurtimox (NFX), show limited efficacy and multiple side effects. Thus, there is a need to develop new trypanocidal strategies. Ivermectin (IVM) is a broad-spectrum antiparasitic drug with low human and veterinary toxicity with effects against T. brucei and Leishmania spp. Considering this and its relatively low cost, we evaluate IVM as a potential repurposed trypanocidal drug on T. cruzi and other trypanosomatids. We found that IVM affected, in a dose-dependent manner, the proliferation of T. cruzi epimastigotes as well as the amastigotes and trypomastigotes survival. The Selectivity Index for the amastigote stage with respect to Vero cells was 12. The IVM effect was also observed in Phytomonas jma 066 and Leishmania mexicana proliferation but not in Crithidia fasciculata. On the epimastigote stage, the IVM effect was trypanostatic at 50 μM but trypanocidal at 100 μM. The assays of the drug combinations of IVM with BNZ or NFX showed mainly additive effects among combinations. In silico studies showed that classical structures belonging to glutamate-gated Cl channels, the most common IVM target, are absent in kinetoplastids. However, we found in the studied trypanosomatid genomes one copy for putative IMPα and IMPβ, potential targets for IVM. The putative IMPα genes (with 76% similarity) showed conserved Armadillo domains but lacked the canonical IMPβ binding sequence. These results allowed us to propose a novel molecular target in T. cruzi and suggest IVM as a good candidate for drug repurposing in the Chagas disease context

Primera detección de Rickettsia asembonensis en Ctenocephalides felis felis en Argentina : Un estudio epidemiológico en pulgas de animales de compañía y sinantrópicos en el trifinio del Noreste argentino

Las rickettsiosis son enfermedades zoonóticas causadas por bacterias del género Rickettsia y transmitidas a humanos por medio de artrópodos vectores. En este sentido, las pulgas son importantes en salud pública debido a su rol como parásitos y como vectores de bacterias patógenas.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

Riboflavin Transporter in Trypanosomatids: Novel Therapeutic Target

La riboflavina (vitamina B2) es un nutriente esencial para todo tipo celular estudiado, siendo elprecursor de los cofactores flavin mononucleótido (FMN) y flavin adenin dinucleótido (FAD). Losmetazoos, incluyendo al humano, carecen de la vía biosintética de riboflavina y la obtienen de ladieta a través de transportadores específicos (familia SLC52). Muchos patógenos sintetizanriboflavina de novo mientras otros la incorporan a través de transportadores específicos,estructuralmente diferentes de los transportadores presentes en los metazoos. Por laesencialidad de la riboflavina y las diferencias entre patógenos y hospedadores, las vías deobtención de riboflavina fueron propuestas como potenciales blancos terapéuticos contra lospatógenos. Los tripanosomátidos son un grupo monofilético comprendiendo parásitos responsables degraves enfermedades en humanos tales como Trypanosoma cruzi (Enfermedad de Chagas), Trypanosoma brucei (Tripanosomiasis Humana Africana) y Leishmania spp. (Leishmaniasiscutánea, mucocutánea y visceral). Otros tripanosomátidos son fitoparásitos, como Phytomonasspp., generando grandes pérdidas en economías regionales de Sudamérica. En esta tesis se estudió metabolismo (posibles rutas de obtención por biosíntesis y/o transporte)de riboflavina en tripanosomátidos así como su rol biológico en los distintos estadíos celulares. Nuestros resultados mostraron que las flavinas son esenciales para la proliferación detripanosomátidos. La riboflavina es incorporada a través de un mecanismo mediado por un/ostransportador/es saturable/s. Más aún, procesos biológicos de T. cruzi como la metaciclogénesisy replicación de epimastigotes y de amastigotes en células de mamíferos son dependientes deltransporte de riboflavina. Mediante análisis bioinformáticos, identificamos una nueva familia detransportadores de riboflavina a la que llamamos “RibJ” y caracterizamos funcionalmente lostransportadores RibJ de T. cruzi y T. brucei. Interesantemente, los análisis filogenéticos mostraronque RibJ es específica para tripanosomátidos y no está relacionada con ninguna de las 3 familiasdescriptas en eucariotas. Dado que riboflavina es esencial para tripanosomátidos, que se obtienepor transporte y que RibJ es estructuralmente distinto a los transportadores de otros eucariotas, incluidos los hospedadores, RibJ puede postularse como nuevo blanco para el diseño de drogascon potencial uso en el desarrollo de terapias tripanocidas.Riboflavin (vitamin B2) is an essential nutrient for all studied living cells, being the precursor offlavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. Metazoa, includinghumans, lack the riboflavin biosynthetic pathway and obtain this vitamin from the diet throughits riboflavin transporter (SLC52 family). Many pathogens synthesize riboflavin de novo whileothers incorporate it through specific riboflavin transporters, structurally different from thosepresent in metazoans. For these reasons, the riboflavin pathways have been proposed astherapeutic targets against pathogens. Trypanosomatids are a monophyletic group encompassing parasites responsible of serioushuman diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Human African Trypanosomiasis) and Leishmania spp. (cutaneous, mucocutaneous, and visceralleishmaniasis). Other ones are phytoparasites, as Phytomonasspp., generating regional economiclosses in South America. In this Thesis, we have studied the metabolism (biosynthesis and transport) of riboflavin intrypanosomatids, defining the biological role of this vitamin at the different cellular stages. Ourresults show that flavins are essential for trypanosomatids proliferation, and that riboflavin isincorporated through a saturable carrier-mediated process. Moreover, we have found thatparasites proliferation and T. cruzi metacyclogenesis and amastigote replication in mammaliancells are dependent on flavin transport. By bioinformatics analysis, we have identified a novelfamily of riboflavin transporters, which we named RibJ, functionally characterizing two relevantmembers from this family: TcRibJ and TbRibJ from T. cruzi and T. brucei, respectively. Interestingly, the phylogenetic analysis show that RibJ is specific for trypanosomatids and it is notrelated with the other three families described in eukaryotic cells. This work highlights theessentiality of riboflavin metabolism for trypanosomatids and presents RibJ as an attractive targetfor drug design. Specifically blockage of riboflavin transport may lead to novel therapeuticsagainst trypanosomiasis.Fil: Balcazar, Darío E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina