Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.FAPESPFAPESPCNPqCNP

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

Abstract In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg 2 L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg 2 mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosoltargeted ARG lacking the glycosomal SKL targeting sequence (argDSKL) restored growth but failed to restore infectivity. Further study showed that the ARGDSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg− L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg− mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration

The relationship between the cellular location of Leishmania (Leishmania) amazonensis arginase and its role during murine macrophage infection

Nos hospedeiros mamíferos, os parasitas do gênero Leishmania vivem nos macrófagos se evadindo de mecanismos microbicidas dessas células, tais como a produção de óxido nítrico (NO). A produção de NO pela enzima óxido nítrico sintase induzida (iNOS) nos macrófagos requer L-arginina como substrato, o mesmo aminoácido utilizado pela arginase para produzir ornitina e uréia. Logo, a arginase pode atuar na sobrevivência de Leishmania no hospedeiro competindo com a iNOS, reduzindo a produção de NO, além de seu papel na via de poliaminas, essencial para a replicação dessas células. Com isso, o objetivo desse estudo é elucidar o papel da arginase de L. (L.) amazonensis durante o ciclo de vida do parasita, particularmente, sua função no estabelecimento e na manutenção da infecção da célula hospedeira, e como esse papel seria exercido. Nesse sentido, obtivemos soros policlonais anti-arginase, a partir da arginase recombinante de L. (L.) amazonensis purificada, e esses soros foram utilizados na imunomarcação da enzima em preparações com formas promastigotas e macrófagos infectados com amastigotas de L. (L.) amazonensis. Assim, determinamos a compartimentalização da arginase nos glicossomos tanto na forma promastigota do parasita como na forma amastigota, durante a infecção. Além disso, obtivemos diversos mutantes com a expressão de arginase modificada quanto à quantidade e localização que nos permitiram avaliar a importância da compartimentalização dessa enzima nos glicossomos. Entre esses mutantes temos: superexpressores de arginase, com e sem sinal de endereçamento para glicossomo; parasitas com um alelo de arginase nocauteado e o outro substituído pelo cassete contendo o segmento ddFKBP-ARG, que teriam a expressão de arginase regulada pelo domínio ddFKB sendo nocautes funcionais de arginase; e finalmente, também obtivemos parasitas nocaute nulo de arginase. A análise desses mutantes permitiu conclusões importantes para o conhecimento da fisiologia do parasita e sua relação com o macrófago, revelando que o papel da arginase de Leishmania parece ser muito mais complexo do que o inicialmente postulado, participando na regulação de outras vias metabólicas do próprio parasita e da célula hospedeira. Paralelamente, também determinamos que o sistema ddFKBP é funcional em L. (L.) amazonensis, e assim pode ser utilizado no estudo funcional de outras proteínas importantes para esses parasitas.In the mammal host, Leishmania parasites live inside macrophages escaping from their microbicidal mechanisms, such as the nitric oxide (NO) production. The macrophage NO production by inducible nitric oxide synthase (iNOS) requires L-arginine as substrate, the same amino acid required by arginase to generate ornithine and urea. So, arginase may play a dual role in Leishmania survival reducing the NO by competing with iNOS, and participating in the polyamines pathway, which is essential for the cells replication. Considering this, the aim of this study is to elucidate the role of L. (L.) amazonensis arginase during the parasite life cycle, mainly its function for the establishment and maintenance of the host cell infection, besides to elucidate the way that this enzyme plays its role. With this in mind, we obtained polyclonal anti-arginase sera using purified recombinant L. (L.) amazonensis arginase, these sera were used in immunolabelling assays of L. (L.) amazonensis promastigotes and macrophages infected with L. (L.) amazonensis amastigotes. These experiments determined that arginase is compartmentalized in the glycosomes of both promastigotes and amastigotes, during infection. Besides, we obtained several mutants with altered arginase expression, modified in terms of quantity and location, which permitted us to evaluate the importance of glycosome arginase compartmentalization. Among these mutants are: overexpressors of arginase, with and without glycosomal addressing signal; parasites with one arginase allele knocked out and the other one replaced by a sequence containing the ddFKBP-ARG fusion that would allow us to regulate arginase expression, working like a functional arginase knockout; and finally, we also obtained arginase null knockouts parasites. The mutants analyses lead us to important conclusions for the knowledge of the parasite physiology and its relationship with the host macrophage, revealing that the Leishmania arginase role appears to be more complex than previously thought, playing an important role in the regulation of other metabolic pathways, of the own parasite and of the host cell. In the other hand, we also determined that the ddFKBP system is functional in L. (L.) amazonensis, and then can be used for functional studies of other important parasite´s proteins

One Health Approach to Leishmaniases: Understanding the Disease Dynamics through Diagnostic Tools

Leishmaniases are zoonotic vector-borne diseases caused by protozoan parasites of the genus Leishmania that affect millions of people around the globe. There are various clinical manifestations, ranging from self-healing cutaneous lesions to potentially fatal visceral leishmaniasis, all of which are associated with different Leishmania species. Transmission of these parasites is complex due to the varying ecological relationships between human and/or animal reservoir hosts, parasites, and sand fly vectors. Moreover, vector-borne diseases like leishmaniases are intricately linked to environmental changes and socioeconomic risk factors, advocating the importance of the One Health approach to control these diseases. The development of an accurate, fast, and cost-effective diagnostic tool for leishmaniases is a priority, and the implementation of various control measures such as animal sentinel surveillance systems is needed to better detect, prevent, and respond to the (re-)emergence of leishmaniases

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253 +/- 0.01 mM and a maximum velocity of 0.246 +/- 0.04 nmol/min per 107 cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host. (C) 2008 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

The iron-dependent mitochondrial superoxide dismutase SODA promotesLeishmania virulence

Menezes, Juliana Perrone Bezerra de. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-26T14:14:29Z No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-26T14:34:09Z (GMT) No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5)Made available in DSpace on 2018-03-26T14:34:09Z (GMT). No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5) Previous issue date: 2017National Institutes of Health Grant R01 AI067979 (to N. W. A.).University of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USALeishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide.Leishmaniainfection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role,Leishmaniamitochondrial SOD may also initiate H2O2-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform inLeishmania amazonensisOur inability to generateL. amazonensis SODAnull mutants and the lethal phenotype observed following RNAi-mediated silencing of theTrypanosoma brucei SODAortholog suggests that SODA is essential for trypanosomatid survival.L. amazonensismetacyclic promastigotes lacking oneSODAallele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure ofSODA/ΔsodApromastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, asSODA/ΔsodAcultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotesLeishmaniavirulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms