RESUMO
The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.
Assuntos
Doença de Chagas/parasitologia , Mitocôndrias/metabolismo , Prolina Oxidase/metabolismo , Rhodnius/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Diferenciação CelularRESUMO
Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.
Assuntos
Núcleo Celular/metabolismo , Estágios do Ciclo de Vida , Morfogênese , Proteína de Replicação A/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Doença de Chagas/sangue , Doença de Chagas/parasitologia , Simulação por Computador , Citoplasma/metabolismo , Morfogênese/genética , Sinais de Exportação Nuclear/genética , Sinais de Exportação Nuclear/fisiologia , Proteína de Replicação A/genética , Trypanosoma cruzi/citologiaRESUMO
DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.
Assuntos
Cromatina/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Cromatina/genética , DNA Helicases/genética , DNA Polimerase Dirigida por DNA/genética , Humanos , Complexo de Reconhecimento de Origem/genética , Proteínas de Protozoários/genética , Origem de Replicação , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , DNA Polimerase tetaRESUMO
Here, we investigated the features of replication in Trypanosoma cruzi epimastigotes based on fork speed progression, which is influenced by distinct features such as DNA polymerase rate, susceptibility to DNA damage and repair, secondary structures, transcription and chromatin state. Although T. cruzi exhibits a mean fork speed (2.05 ± 0.10 kb/min) very similar to other trypanosomatids, we found that the majority of DNA molecules replicated more slowly, with a frequency distribution approximately 1 kb/min. This frequency distribution analysis provides more information about the replication profile of this organism.
Assuntos
Replicação do DNA , DNA de Protozoário/genética , Trypanosoma cruzi/genética , Imagem Individual de MoléculaRESUMO
Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs-i.e., origin distribution, termination site location, and fork rate and direction-of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.