RESUMO
Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.
RESUMO
BACKGROUND: Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion. RESULTS: We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains. CONCLUSIONS: Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.
Assuntos
Entamoeba/citologia , Entamoeba/genética , Genoma de Protozoário/genética , Modelos Genéticos , Óvulo/metabolismo , Parasitos/genética , Transcriptoma/genética , Animais , Northern Blotting , Forma Celular/genética , DNA Intergênico/genética , Bases de Dados de Proteínas , Éxons/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Íntrons/genética , Fosfolipase D/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Estatística como Assunto , Fatores de TempoRESUMO
VIPER was initially characterized as a 2326bp LTR-like retroelement associated to SIRE, a short interspersed repetitive element specific of Trypanosoma cruzi. It carried a single ORF that coded for a putative reverse transcriptase-RNAse H protein, suggesting that it could be a truncated copy of a longer retroelement. Herein we report the identification and characterization of a complete 4480bp long VIPER in the T. cruzi genome. The complete VIPER harbored three non-overlapped domains encoding for a GAG-like, a tyrosine recombinase and a reverse transcriptase-RNAse H proteins. VIPER elements were also found in the genomes of Trypanosoma brucei and Trypanosoma vivax, but not in Leishmania sp. On the basis of its reverse transcriptase phylogeny, VIPER was classified as an LTR retroelement. However, VIPER was structurally related to the tyrosine recombinase encoding retroelements, DIRS and Ngaro. Phylogenetic analysis showed that VIPER's tyrosine recombinase grouped with the transposases RCI1 of Escherichia coli and Ye24 and Ye72 of Haemophilus influenzae within a major branch of prokaryotic recombinases. Taken together, VIPER's structure, the nature of its tyrosine recombinase, the unique features of its reverse transcriptase catalytic consensus motif and the fact that it was found in Trypanosomes, an early branching eukaryote, suggest that VIPER may be the closest relative of the founder element of the tyrosine recombinase encoding retrotransposons known up to date. Our analysis revealed that tyrosine recombinase-encoding retroelements were originated as early in evolution as non-LTR retroelements and suggests that VIPER, Ngaro and DIRS elements may constitute a third group of retrotransposons, distinct from both LTR and non-LTR retroelements.