RESUMO
Four anatomical patterns of hydrocephalus secondary to congenital Toxoplasma gondii infection were identified and characterized for infants enrolled in the National Collaborative Chicago-based Congenital Toxoplasmosis Study. Analysis of parasite serotype revealed that different anatomical patterns associate with Type-II vs Not-Exclusively Type-II strains (NE-II) (P = .035).
Assuntos
Genótipo , Hidrocefalia/patologia , Hidrocefalia/parasitologia , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose Congênita/complicações , Estudos de Coortes , Humanos , Sorogrupo , Toxoplasma/isolamento & purificaçãoRESUMO
Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.
Assuntos
Ciclo Celular , Proliferação de Células , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , Proteínas Ribossômicas/genética , Toxoplasma/genética , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Western Blotting , Encéfalo/metabolismo , Encéfalo/parasitologia , Encéfalo/patologia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Imunização , Camundongos , Modelos Genéticos , Ligação Proteica , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/imunologia , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/genética , Tetraciclinas/farmacologia , Toxoplasma/imunologia , Fatores de Transcrição/metabolismoRESUMO
Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC(90)s < or = 6 microM without toxicity to host cells, three compounds have IC(90)s < 45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.
