RESUMEN

Trypanosoma cruzi, the etiological agent of Chagas' disease, can infect both phagocytic and non-phagocytic cells. T. cruzi gp82 and gp90 are cell surface proteins belonging to Group II trans-sialidases known to be involved in host cell binding and invasion. Phosphatidylinositol kinases (PIK) are lipid kinases that phosphorylate phospholipids in their substrates or in themselves, regulating important cellular functions such as metabolism, cell cycle and survival. Vps34, a class III PIK, regulates autophagy, trimeric G-protein signaling, and the mTOR (mammalian Target of Rapamycin) nutrient-sensing pathway. The mammalian autophagy gene Beclin1 interacts to Vps34 forming Beclin 1-Vps34 complexes involved in autophagy and protein sorting. In T. cruzi epimastigotes, (a non-infective replicative form), TcVps34 has been related to morphological and functional changes associated to vesicular trafficking, osmoregulation and receptor-mediated endocytosis. We aimed to characterize the role of TcVps34 during invasion of HeLa cells by metacyclic (MT) forms. MTs overexpressing TcVps34 showed lower invasion rates compared to controls, whilst exhibiting a significant decrease in gp82 expression in the parasite surface. In addition, we showed that T. cruzi Beclin (TcBeclin1) colocalizes with TcVps34 in epimastigotes, thus suggesting the formation of complexes that may play conserved cellular roles already described for other eukaryotes.

RESUMEN

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

Asunto(s)

Enfermedad de Chagas , Proteínas Protozoarias , Trypanosoma cruzi , Animales , Humanos , Ratones , Línea Celular , Enfermedad de Chagas/parasitología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Interacciones Huésped-Parásitos , Ratones Endogámicos BALB C , Parasitemia , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/metabolismo , Virulencia

RESUMEN

Trypanosomatids are flagellate protozoans that can infect several invertebrate and vertebrate hosts, including insects and humans. The three most studied species are the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. which are the causative agents of Human African Trypanosomiasis (HAT), Chagas disease and different clinical forms of leishmaniasis, respectively. These parasites possess complex dixenous life cycles, with zoonotic and anthroponotic stages, and are transmitted by hematophagous insects. To colonize this myriad of hosts, they developed mechanisms, mediated by virulence factors, to infect, propagate and survive in different environments. In insects, surface proteins play roles in parasite attachment and survival in the insect gut, whilst in the mammalian host, the parasites have a whole group of proteins and mechanisms that aid them invading the host cells and evading its immune system components. Many studies have been done on the impact of these molecules in the vertebrate host, however it is also essential to notice the importance of these virulence factors in the insect vector during the parasite life cycle. When inside the insect, the parasites, like in humans, also need to survive defense mechanisms components that can inhibit parasite colonization or survival, e.g., midgut peritrophic membrane barrier, digestive enzymes, evasion of excretion alongside the digested blood meal, anatomic structures and physiological mechanisms of the anterior gut. This protection inside the insect is often implemented by the same group of virulence factors that perform roles of immune evasion in the mammalian host with just a few exceptions, in which a specific protein is expressed specifically for the insect vector form of the parasite. This review aims to discuss the roles of the virulence molecules in the insect vectors, showing the differences and similarities of modes of action of the same group of molecules in insect and humans, exclusive insect molecules and discuss possible genetic events that may have generated this protein diversity.

Asunto(s)

Enfermedad de Chagas , Parásitos , Trypanosoma cruzi , Animales , Enfermedad de Chagas/parasitología , Humanos , Insectos Vectores/parasitología , Insectos , Mamíferos , Proteínas de la Membrana , Trypanosoma cruzi/fisiología , Factores de Virulencia/genética

RESUMEN

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits extensive inter- and intrastrain genetic diversity. As we have previously described, there are some genetic differences between the parental G strain and its clone D11, which was isolated by the limiting dilution method and infection of cultured mammalian cells. Electrophoretic karyotyping and Southern blot hybridization of chromosomal bands with specific markers revealed chromosome length polymorphisms of small size with additional chromosomal bands in clone D11 and the maintenance of large syntenic groups. Both G strain and clone D11 belong to the T. cruzi lineage TcI. Here, we designed intraspecific array-based comparative genomic hybridization (aCGH) to identify chromosomal regions harboring copy-number variations between clone D11 and the G strain. DNA losses were more extensive than DNA gains in clone D11. Most alterations were flanked by repeated sequences from multigene families that could be involved in the duplication and deletion events. Several rearrangements were detected by chromoblot hybridization and confirmed by aCGH. We have integrated the information of genomic sequence data obtained by aCGH to the electrophoretic karyotype, allowing the reconstruction of possible recombination events that could have generated the karyotype of clone D11. These rearrangements may be explained by unequal crossing over between sister or homologous chromatids mediated by flanking repeated sequences and unequal homologous recombination via break-induced replication. The genomic changes detected by aCGH suggest the presence of a dynamic genome that responds to environmental stress by varying the number of gene copies and generating segmental aneuploidy.

Asunto(s)

Enfermedad de Chagas , Trypanosoma cruzi , Animales , Células Clonales , Hibridación Genómica Comparativa/métodos , ADN , Genoma de Protozoos , Mamíferos/genética , Trypanosoma cruzi/genética

RESUMEN

P21 is an immunomodulatory protein expressed throughout the life cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease. In vitro and in vivo studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization. Furthermore, in vivo studies have shown that P21 inhibits angiogenesis, induces inflammation and fibrosis, and regulates intracellular amastigote replication. In this study, we used the CRISPR/Cas9 system for P21 gene knockout and investigated whether the ablation of P21 results in changes in the phenotypes associated with this protein. Ablation of P21 gene resulted in a lower growth rate of epimastigotes and delayed cell cycle progression, accompanied by accumulation of parasites in G1 phase. However, P21 knockout epimastigotes were viable and able to differentiate into metacyclic trypomastigotes, which are infective to mammalian cells. In comparison with wild-type parasites, P21 knockout cells showed a reduced cell invasion rate, demonstrating the role of this protein in host cell invasion. However, there was a higher number of intracellular amastigotes per cell, suggesting that P21 is a negative regulator of amastigote proliferation in mammalian cells. Here, for the first time, we demonstrated the direct correlation between P21 and the replication of intracellular amastigotes, which underlies the chronicity of T. cruzi infection.

Asunto(s)

Enfermedad de Chagas , Trypanosoma cruzi , Citoesqueleto de Actina/fisiología , Animales , Enfermedad de Chagas/parasitología , Técnicas de Inactivación de Genes , Estadios del Ciclo de Vida/fisiología , Mamíferos/genética , Ratones , Trypanosoma cruzi/fisiología

RESUMEN

Trypanosoma brucei, Leishmania spp., and T. cruzi are flagellate protozoans of the family Trypanosomatidae and the causative agents of human African trypanosomiasis, leishmaniasis, and Chagas disease, respectively. These diseases affect humans worldwide and exert a significant impact on public health. Over the course of evolution, the parasites associated with these pathologies have developed mechanisms to circumvent the immune response system throughout the infection cycle. In cases of human infection, this function is undertaken by a group of proteins and processes that allow the parasites to propagate and survive during host invasion. In T. brucei, antigenic variation is promoted by variant surface glycoproteins and other proteins involved in evasion from the humoral immune response, which helps the parasite sustain itself in the extracellular milieu during infection. Conversely, Leishmania spp. and T. cruzi possess a more complex infection cycle, with specific intracellular stages. In addition to mechanisms for evading humoral immunity, the pathogens have also developed mechanisms for facilitating their adhesion and incorporation into host cells. In this review, the different immune evasion strategies at cellular and molecular levels developed by these human-pathogenic trypanosomatids have been discussed, with a focus on the key molecules responsible for mediating the invasion and evasion mechanisms and the effects of these molecules on virulence.

Asunto(s)

Enfermedad de Chagas , Leishmaniasis , Trypanosoma brucei brucei , Trypanosoma cruzi , Humanos , Virulencia

RESUMEN

Trypanosoma cruzi is the etiological agent of Chagas disease, which affects millions of people in Latin America. No transcriptional control of gene expression has been demonstrated in this organism, and 50% of its genome consists of repetitive elements and members of multigenic families. In this study, we applied a novel bioinformatics approach to predict new repetitive elements in the genome sequence of T. cruzi. A new repetitive sequence measuring 241 nt was identified and found to be interspersed along the genome sequence from strains of different DTUs. This new repeat was mostly on intergenic regions, and upstream and downstream regions of the 241 nt repeat were enriched in surface protein genes. RNAseq analysis revealed that the repeat was part of processed mRNAs and was predominantly found in the 3' untranslated regions (UTRs) of genes of multigenic families encoding surface proteins. Moreover, we detected a correlation between the presence of the repeat in the 3'UTR of multigenic family genes and the level of differential expression of these genes when comparing epimastigote and trypomastigote transcriptomes. These data suggest that this sequence plays a role in the posttranscriptional regulation of the expression of multigenic families.

Asunto(s)

Regiones no Traducidas 3'/genética , Genoma de Protozoos , Secuencias Repetitivas Esparcidas , Proteínas de la Membrana/metabolismo , Familia de Multigenes , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Enfermedad de Chagas/genética , Enfermedad de Chagas/parasitología , ADN Protozoario/análisis , ADN Protozoario/genética , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Análisis de Secuencia de ADN , Trypanosoma cruzi/metabolismo

RESUMEN

Chagas disease, a zoonosis caused by the flagellate protozoan Trypanosoma cruzi, is a chronic and systemic parasitic infection that affects ~5-7 million people worldwide, mainly in Latin America. Chagas disease is an emerging public health problem due to the lack of vaccines and effective treatments. According to recent studies, several T. cruzi secreted proteins interact with the human host during cell invasion. Moreover, some comparative studies with T. rangeli, which is non-pathogenic in humans, have been performed to identify proteins directly involved in the pathogenesis of the disease. In this study, we present an integrated analysis of canonical putative secreted proteins (PSPs) from both species. Additionally, we propose an interactome with human host and gene family clusters, and a phylogenetic inference of a selected protein. In total, we identified 322 exclusively PSPs in T. cruzi and 202 in T. rangeli. Among the PSPs identified in T. cruzi, we found several trans-sialidases, mucins, MASPs, proteins with phospholipase 2 domains (PLA2-like), and proteins with Hsp70 domains (Hsp70-like) which have been previously characterized and demonstrated to be related to T. cruzi virulence. PSPs found in T. rangeli were related to protozoan metabolism, specifically carboxylases and phosphatases. Furthermore, we also identified PSPs that may interact with the human immune system, including heat shock and MASP proteins, but in a lower number compared to T. cruzi. Interestingly, we describe a hypothetical hybrid interactome of PSPs which reveals that T. cruzi secreted molecules may be down-regulating IL-17 whilst T. rangeli may enhance the production of IL-15. These results will pave the way for a better understanding of the pathophysiology of Chagas disease and may ultimately lead to the identification of molecular targets, such as key PSPs, that could be used to minimize the health outcomes of Chagas disease by modulating the immune response triggered by T. cruzi infection.

Asunto(s)

Enfermedad de Chagas/parasitología , Proteoma , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma rangeli/metabolismo , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/metabolismo , Biología Computacional , Regulación Viral de la Expresión Génica , Redes Reguladoras de Genes , Genómica , Interacciones Huésped-Patógeno , Humanos , Filogenia , Mapas de Interacción de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Vías Secretoras , Transducción de Señal , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Trypanosoma rangeli/genética , Trypanosoma rangeli/inmunología

RESUMEN

The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatusMSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatusmshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The ΔmshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella Wild-type and ΔmshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the ΔmshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by ΔmshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition.IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2 Although the mutation frequency of mshA (the A. fumigatusMSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.

Asunto(s)

Antifúngicos/farmacología , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidad , Azoles/farmacología , Farmacorresistencia Fúngica , Proteína 2 Homóloga a MutS/genética , Animales , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Reparación de la Incompatibilidad de ADN , Femenino , Proteínas Fúngicas/genética , Larva/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , Neutropenia , Homología de Secuencia , Virulencia

RESUMEN

Trypanosoma cruzi, the causative agent of Chagas disease, has a dense coat of GPI-anchored virulence factors. T. cruzi GPI-anchored adhesin GP82 is encoded by a repertoire of transcripts containing several in-frame initiation codons located up-stream from that adjacent to the predicted signal peptide (SP). Transfection of T. cruzi epimastigotes with constructs encoding GP82 starting at the SP or from the farthest up-stream methionine confirmed protein expression on the parasite cell surface, comparable to the native GP82. Proteins were fully functional, inducing parasite adhesion to HeLa cells and lysosome mobilization, events required for parasite invasion. Transgenic and native GP82 proteins showed indistinguishable electrophoretic mobility, suggesting similar processing of the SP. Deletion of SP generated a ~72 kDa protein devoid of N-linked oligosaccharides allowing irrefutable identification of GP82 precursor. SP transposition to an internal region of GP82 rendered the signal unrecognizable by the signal peptidase and incapable to direct the nascent protein for ER-membrane association. Altogether our data strongly suggests that GP82 SP fails to function as transmembrane domain and its recognition by the signal peptidase shows strict dependence on the signal localization at protein N-terminus. This report presents the first experimental characterization of the full-length GP82 and its signal peptide.

Asunto(s)

Enfermedad de Chagas/patología , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/patogenicidad , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Factores de Virulencia/metabolismo , Enfermedad de Chagas/parasitología , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Protozoarias/genética , Alineación de Secuencia , Relación Estructura-Actividad , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Factores de Virulencia/genética

RESUMEN

Genetic stability is extremely important for the survival of every living organism, and a very complex set of genes has evolved to cope with DNA repair upon DNA damage. Here, we investigated the Aspergillus fumigatus AtmA (Ataxia-telangiectasia mutated, ATM) and AtrA kinases, and how they impact virulence and the evolution of azole resistance. We demonstrated that A. fumigatus atmA and atrA null mutants are haploid and have a discrete chromosomal polymorphism. The ΔatmA and ΔatrA strains are sensitive to several DNA-damaging agents, but surprisingly both strains were more resistant than the wild-type strain to paraquat, menadione, and hydrogen peroxide. The atmA and atrA genes showed synthetic lethality emphasizing the cooperation between both enzymes and their consequent redundancy. The lack of atmA and atrA does not cause any significant virulence reduction in A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonela Wild-type, ΔatmA, and ΔatrA populations that were previously transferred 10 times in minimal medium (MM) in the absence of voriconazole have not shown any significant changes in drug resistance acquisition. In contrast, ΔatmA and ΔatrA populations that similarly evolved in the presence of a subinhibitory concentration of voriconazole showed an ∼5-10-fold increase when compared to the original minimal inhibitory concentration (MIC) values. There are discrete alterations in the voriconazole target Cyp51A/Erg11A or cyp51/erg11 and/or Cdr1B efflux transporter overexpression that do not seem to be the main mechanisms to explain voriconazole resistance in these evolved populations. Taken together, these results suggest that genetic instability caused by ΔatmA and ΔatrA mutations can confer an adaptive advantage, mainly in the intensity of voriconazole resistance acquisition.

Asunto(s)

Aspergillus fumigatus/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Farmacorresistencia Fúngica/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Voriconazol/farmacología , Animales , Antifúngicos/farmacología , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inestabilidad Genómica , Humanos , Aspergilosis Pulmonar Invasiva/tratamiento farmacológico , Aspergilosis Pulmonar Invasiva/microbiología , Aspergilosis Pulmonar Invasiva/mortalidad , Aspergilosis Pulmonar Invasiva/patología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , Mutación , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Análisis de Supervivencia , Virulencia

RESUMEN

Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion.

Asunto(s)

Interacciones Huésped-Parásitos/fisiología , Microcuerpos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Trypanosoma cruzi/enzimología , Citoesqueleto de Actina , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/inmunología , Dimerización , Células HeLa , Humanos , Estadios del Ciclo de Vida , Ratones , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Simulación de Dinámica Molecular , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunología , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Trypanosoma cruzi/fisiología

RESUMEN

BACKGROUND: Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes. RESULTS: We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR. CONCLUSION: Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Genoma Fúngico/genética , Paracoccidioides/genética , Retroelementos/genética , Análisis por Conglomerados , Etiquetas de Secuencia Expresada/metabolismo , Genómica , Anotación de Secuencia Molecular , Paracoccidioides/clasificación , Filogenia , Secuencias Repetidas Terminales/genética , Transcripción Genética

RESUMEN

Chagas disease is caused by the protozoan Trypanosoma cruzi which affects 10 million people worldwide. Very few kinases have been characterized in this parasite, including the phosphatidylinositol kinases (PIKs) that are at the heart of one of the major pathways of intracellular signal transduction. Recently, we have classified the PIK family in T. cruzi using five different models based on the presence of PIK conserved domains. In this study, we have mapped PIK genes to the chromosomes of two different T. cruzi lineages (G and CL Brener) and determined the cellular localization of two PIK members. The kinases have crucial roles in metabolism and are assumed to be conserved throughout evolution. For this reason, they should display a conserved localization within the same eukaryotic species. In spite of this, there is an extensive polymorphism regarding PIK localization at both genomic and cellular levels, among different T. cruzi isolates and between T. cruzi and Trypanosomabrucei, respectively. We showed in this study that the cellular localization of two PIK-related proteins (TOR1 and 2) in the T. cruzi lineage is distinct from that previously observed in T. brucei. In addition, we identified a new PIK gene with peculiar feature, that is, it codes for a FYVE domain at N-terminal position. FYVE-PIK genes are phylogenetically distant from the groups containing exclusively the FYVE or PIK domain. The FYVE-PIK architecture is only present in trypanosomatids and in virus such as Acanthamoeba mimivirus, suggesting a horizontal acquisition. Our Bayesian phylogenetic inference supports this hypothesis. The exact functions of this FYVE-PIK gene are unknown, but the presence of FYVE domain suggests a role in membranous compartments, such as endosome. Taken together, the data presented here strengthen the possibility that trypanosomatids are characterized by extensive genomic plasticity that may be considered in designing drugs and vaccines for prevention of Chagas disease.

Asunto(s)

Fosfatidilinositol 3-Quinasas/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/enzimología , Teorema de Bayes , Enfermedad de Chagas/epidemiología , Evolución Molecular , Transferencia de Gen Horizontal , Genoma de Protozoos , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Filogenia , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética

RESUMEN

The identification of new targets for vaccine and drug development for the treatment of Chagas' disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite's genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6-8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.

Asunto(s)

Clonación Molecular , Regulación de la Expresión Génica/fisiología , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Animales , Cromosomas Artificiales , Ratones , Organismos Modificados Genéticamente

RESUMEN

Parasitological cure for Chagas disease is considered extremely difficult to achieve because of the lack of effective chemotherapeutic agents against Trypanosoma cruzi at different stages of infection. There are currently only two drugs available. These have several limitations and can produce serious side effects. Thus, new chemotherapeutic targets are much sought after. Among T. cruzi components involved in key processes such as parasite proliferation and host cell invasion, Ca(2+)-dependent molecules play an important role. Calcineurin (CaN) is one such molecule. In this study, we cloned a new isoform of the gene coding for CL strain catalytic subunit CaNA (TcCaNA2) and characterized it molecularly and functionally. There is one copy of the TcCaNA2 gene per haploid genome. It is constitutively transcribed in all T. cruzi developmental forms and is localized predominantly in the cytosol. In the parasite, TcCaNA2 is associated with CaNB. The recombinant protein TcCaNA2 has phosphatase activity that is enhanced by Mn(2+)/Ni(2+). The participation of TcCaNA2 in target cell invasion by metacyclic trypomastigotes was also demonstrated. Metacyclic forms with reduced TcCaNA2 expression following treatment with morpholino antisense oligonucleotides targeted to TcCaNA2 invaded HeLa cells at a lower rate than control parasites treated with morpholino sense oligonucleotides. Similarly, the decreased expression of TcCaNA2 following treatment with antisense morpholino oligonucleotides partially affected the replication of epimastigotes, although to a lesser extent than the decrease in expression following treatment with calcineurin inhibitors. Our findings suggest that the calcineurin activities of TcCaNA2/CaNB and TcCaNA/CaNB, which have distinct cellular localizations (the cytoplasm and the nucleus, respectively), may play a critical role at different stages of T. cruzi development, the former in host cell invasion and the latter in parasite multiplication.

Asunto(s)

Calcineurina/genética , Calcineurina/metabolismo , Trypanosoma cruzi/metabolismo , Antígenos de Protozoos , Dominio Catalítico/genética , Proliferación Celular , Clonación Molecular , Endocitosis , Activadores de Enzimas/metabolismo , Células HeLa , Humanos , Manganeso/metabolismo , Datos de Secuencia Molecular , Níquel/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Multimerización de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Trypanosoma cruzi/genética

RESUMEN

Sporotrichosis is a polymorphic disease caused by a complex of thermodimorphic fungi including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. Humans and animals can acquire the disease through traumatic inoculation of propagules into the subcutaneous tissue. Despite the importance of sporotrichosis as a disease that can take epidemic proportions there are just a few studies dealing with genetic polymorphisms and genomic architecture of these pathogens. The main objective of this study was to investigate chromosomal polymorphisms and genomic organization among different isolates in the S. schenckii complex. We used pulsed field gel electrophoresis (PFGE) to separate chromosomal fragments of isolated DNA, followed by probe hybridization. Nine loci (ß-tubulin, calmodulin, catalase, chitin synthase 1, Internal Transcribed Spacer, Pho85 cyclin-dependent kinase, protein kinase C Ss-2, G protein α subunit and topoisomerase II) were mapped onto chromosomal bands of Brazilian isolates of S. schenckii s. str. and S. brasiliensis. Our results revealed the presence of intra and interspecies polymorphisms in chromosome number and size. The gene hybridization analysis showed that closely related species in phylogenetic analysis had similar genetic organizations, mostly due to identification of synteny groups in chromosomal bands of similar sizes. Our results bring new insights into the genetic diversity and genome organization among pathogenic species in the Sporothrix schenckii complex.

Asunto(s)

Cromosomas Fúngicos/genética , ADN de Hongos/genética , Polimorfismo Genético/genética , Sporothrix/genética , Sporothrix/patogenicidad , Esporotricosis/microbiología , Virulencia/genética , Southern Blotting , Electroforesis en Gel de Campo Pulsado , Marcadores Genéticos , Variación Genética , Cariotipificación , Filogenia , Reacción en Cadena de la Polimerasa , Sporothrix/clasificación , Esporotricosis/genética

RESUMEN

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.

Asunto(s)

Genoma de Protozoos , Leishmania/genética , Interacciones Huésped-Parásitos , Humanos , Leishmania/metabolismo , Leishmaniasis Cutánea/parasitología , Modelos Genéticos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Filogenia

RESUMEN

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

Asunto(s)

Cromosomas/genética , Reordenamiento Génico/genética , Variación Genética , Cariotipificación , Trypanosoma cruzi/citología , Trypanosoma cruzi/genética , Alelos , Animales , Células Clonales , Secuencia Conservada/genética , Sitios Genéticos/genética , Marcadores Genéticos , Tamaño del Genoma/genética , Genoma de Protozoos/genética , Técnicas de Genotipaje , Repeticiones de Microsatélite/genética , Zarigüeyas , Polimorfismo Genético , Sintenía/genética , Telómero/metabolismo , Tubulina (Proteína)/metabolismo

RESUMEN

T. cruzi improves the likelihood of invading or adapting to the host through its capacity to present a large repertoire of surface molecules. The metacyclic stage-specific surface glycoprotein GP82 has been implicated in host cell invasion. GP82 is encoded by multiple genes from the trans-sialidase superfamily. GP82 shows a modular organization, with some variation of N-terminal region flanking a conserved central core where the binding sites to the mammalian cell and gastric mucin are located. The function of GP82 as adhesin in host cell invasion process could expose the protein to an intense conservative and selective pressure. GP82 is a GPI-anchored surface protein, synthesized as a 70 kDa precursor devoid of N-linked sugars. GPI-minus variants accumulate in the ER indicating that GPI anchor acts as a forward transport signal for progressing along the secretory pathway as suggested for T. cruzi mucins. It has been demonstrated that the expression of GP82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization. GP82 mRNAs are mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes. Analysis of transgenic parasites indicates that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.

Asunto(s)

Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Sitios de Unión , Adhesión Celular , Enfermedad de Chagas/parasitología , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Polirribosomas/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad