RESUMO
The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.
Assuntos
Euglenozoários/citologia , Euglenozoários/metabolismo , Peroxissomos/metabolismo , Trypanosoma cruzi/citologia , Aminoácidos/metabolismo , Carbono/metabolismo , Enzimas/metabolismo , Euglenozoários/genética , Gluconeogênese , Microcorpos , Via de Pentose Fosfato , Filogenia , Transdução de Sinais , Trypanosoma cruzi/metabolismoRESUMO
The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.
Assuntos
Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Citoplasma/parasitologia , Pirimidinas/biossíntese , Trypanosoma cruzi/crescimento & desenvolvimento , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Trypanosoma cruzi/genéticaRESUMO
The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Di-Hidro-Orotase/metabolismo , Pirimidinas/biossíntese , Trypanosoma cruzi/enzimologia , ImunoprecipitaçãoRESUMO
We examined whether antibody isotype responses to paramyosin (PM), a vaccine candidate for schistosomiasis, are associated with age-dependent resistance and pathology in liver fibrosis using human sera collected from 139 individuals infected with Schistosoma japonicum in Leyte, The Philippines. We report that IgA and IgG3 responses to PM showed a positive correlation with age and that the epitopes responsible were localized predominantly within the N-terminal half of PM. In addition, the IgG3 response to PM was associated with serum level of procollagen-III-peptide (P-III-P), an indicator of progression of liver fibrosis. These results imply that IgG3 against PM may not only provoke age-dependent resistance to S. japonicum infection but also enhance liver fibrosis. In contrast, levels of IgE to PM and to multiple PM fragments showed a negative correlation with P-III-P level. Thus, in contrast to IgG3, increases in PM-specific IgE may contribute to suppression of liver pathogenesis in schistosomiasis.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Isotipos de Imunoglobulinas/imunologia , Cirrose Hepática/imunologia , Schistosoma japonicum/imunologia , Esquistossomose Japônica/imunologia , Tropomiosina/imunologia , Adolescente , Adulto , Idoso , Animais , Criança , Estudos de Coortes , Colágeno Tipo IV/imunologia , Epitopos/imunologia , Feminino , Humanos , Cirrose Hepática/parasitologia , Cirrose Hepática/prevenção & controle , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Filipinas , Pró-Colágeno/imunologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esquistossomose Japônica/parasitologia , Esquistossomose Japônica/prevenção & controle , Estatísticas não Paramétricas , Tropomiosina/genéticaRESUMO
Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.
Assuntos
Variação Genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Di-Hidro-Orotato Desidrogenase , Fumaratos/metabolismo , Concentração de Íons de Hidrogênio , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/biossíntese , Mutação Puntual/genética , Polimorfismo Genético , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , TemperaturaRESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 microg/ml. Inhibition by these extracts was noncompetitive with respect to dihydroorotate, with apparent Ki values of 35.3+/-5.9 and 10.3+/-4.4 microg/ml, respectively. Further, in an in vitro T. cruzi-HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 microg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.
Assuntos
Eucariotos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Trypanosoma cruzi/enzimologia , Animais , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacologia , Fucus/química , Células HeLa , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Phaeophyceae/química , Água do Mar/microbiologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidadeRESUMO
Herein, we determined the seroprevalence, seroconversion, and risk factors associated with Toxoplasma gondii (T. gondii) infection among pregnant women in Taipei, Taiwan. Pregnant women attending antenatal consultation in a Taipei medical center were invited, and 104 women completed a self-administered structured questionnaire. Venous blood samples were collected during the first and third trimester after consent was obtained. Serum IgG and IgM antibodies (Abs) as well as IgG avidity were analyzed using an enzyme-linked fluorescent assay. Of the samples collected in the first trimester, seven were seropositive for IgG Abs and one was seropositive for IgG + IgM Abs with a borderline avidity index, resulting in an overall seroprevalence of 7.7%. No statistically significant association was found between toxoplasmosis and age, pregnancy history, or any risk factors. Seroconversion was not detected from paired sera between the first and third trimesters. Pregnant women with senior high school education level or those who claimed to knowing Toxoplasma exhibited a significantly higher seroprevalence than those with bachelor degree (P = 0.05) or those who claimed not to have this knowledge (P = 0.05). Therefore, failure to understand the importance of T. gondii infection and the prevention measures resulted in the development of toxoplasmosis among these women.
Assuntos
Anticorpos Antiprotozoários/sangue , Complicações Infecciosas na Gravidez/epidemiologia , Toxoplasma/imunologia , Toxoplasmose/epidemiologia , Adulto , Afinidade de Anticorpos , Estudos Transversais , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Gravidez , Fatores de Risco , Soroconversão , Estudos Soroepidemiológicos , Inquéritos e Questionários , Taiwan/epidemiologia , Adulto JovemRESUMO
BACKGROUND: The status of Toxoplasma gondii infection among primary schoolchildren (PSC) of the Democratic Republic of São Tomé and Príncipe (DRSTP), West Africa, remains unknown to date. METHODS: A serologic survey and risk factors associated T. gondii infection among PSC in the DRSTP was assessed by the latex agglutination (LA) test and a questionnaire interview including parents' occupation, various uncomfortable symptoms, histories of eating raw or undercooked food, drinking unboiled water, and raising pets, was conducted in October 2010. Schoolchildren from 4 primary schools located in the capital areas were selected, in total 255 serum samples were obtained by venipuncture, of which 123 serum samples were obtained from boys (9.8 ± 1.4 yrs) and 132 serum samples were obtained from girls (9.7 ± 1.3 yrs). RESULTS: The overall seroprevalence of T. gondii infection was 63.1% (161/255). No significant gender difference in seroprevalence was found between boys (62.6%, 77/123) and girls (63.6%, 84/132) (p = 0.9). The older age group of 10 years had insignificantly higher seroprevalence (69.9%, 58/83) than that of the younger age group of 8 year olds (67.7%, 21/31) (p = 0.8). It was noteworthy that the majority of seropositive PSC (75.8%, 122/161) had high LA titers of ≥1: 1024, indirectly indicating acute or repeated Toxoplasma infection. Parents whose jobs were non-skilled workers (73.1%) showed significantly higher seroprevalence than that of semiskilled- (53.9%) or skilled workers (48.8%) (p < 0.05). Children who had a history of raising cats also showed significantly higher seroprevalence than those who did not (p < 0.001).Children who claimed to have had recent ocular manifestation or headache, i.e. within 1 month, seemed to have insignificantly higher seroprevalence than those who did not (p > 0.05). CONCLUSIONS: Parents' educational level and cats kept indoors seemed to be the high risk factors for PSC in acquisition of T. gondii infection. While, ocular manifestation and/or headache of PSC should be checked for the possibility of being T. gondii elicited. Measures such as improving environmental hygiene and intensive educational intervention to both PSC and their parents should be performed immediately so as to reduce T. gondii infection of DRSTP inhabitants including PSC and adults.
Assuntos
Toxoplasma , Toxoplasmose/epidemiologia , Anticorpos Antiprotozoários/sangue , Ilhas Atlânticas/epidemiologia , Criança , Feminino , Humanos , Masculino , Razão de Chances , Fatores de Risco , Estudos SoroepidemiológicosRESUMO
A parasitological survey of Schistosoma haematobium infection among primary schoolchildren in the remote areas of Hhohho and Manzini Provinces in northwestern Swaziland was undertaken. Presence of infection in subjects was confirmed on detection of S. haematobium ova in urine or the presence of hematuria. The intensity of the infection was estimated by calculating the total number of S. haematobium ova present in a 10-ml urine specimen and was expressed in terms of geometric mean intensity (GMI). The prevalence of S. haematobium infection in these populations was 5.3% (21/395) with a GMI of 46.5. Boys had higher prevalence (7.1%, 13/182) and GMI (50.4) than girls (3.8%, 8/213; 40.0) did (P>0.05). Geographically, the prevalence in Manzini schoolchildren (14.6%, 12/82) was significantly higher than that in Hhohho schoolchildren (2.9%, 9/313; P<0.001); however, Hhohho schoolchildren had a higher GMI (70.2) than that observed in Manzini schoolchildren (21.9). Children from schools located in Lowveld had a significantly higher prevalence (11.4%, 19/166) than that in children from schools located in Highveld (0.6%, 1/162) (P<0.0001).
Assuntos
Hematúria/parasitologia , Schistosoma haematobium/patogenicidade , Esquistossomose Urinária/epidemiologia , Urina/parasitologia , Animais , Criança , Essuatíni/epidemiologia , Feminino , Geografia , Hematúria/diagnóstico , Hematúria/epidemiologia , Humanos , Masculino , Óvulo , Prevalência , População Rural , Esquistossomose Urinária/diagnóstico , Esquistossomose Urinária/parasitologia , Esquistossomose Urinária/urina , Instituições Acadêmicas/estatística & dados numéricos , Distribuição por SexoRESUMO
Four monoterpene hydroperoxides were isolated from aerial parts of Chenopodium ambrosioides along with ascaridole (1), the anthelmintic principle of this plant, as anti-trypanosomal compounds. The structures of these monoterpenes were determined to be (-)-(2S,4S)- and (-)-(2R,4S)-p-mentha-1(7),8-dien-2-hydroperoxide (2a and 3a) and (-)-(1R,4S)- and (-)-(1S,4S)-p-mentha-2,8-dien-1-hydroperoxide (4a and 5a) on the basis of spectroscopic methods and chemical correlations. In vitro trypanocidal activities of ascaridole (1) and these hydroperoxides (2a-5a) against epimastigotes of Trypanosoma cruzi were 23, 1.2, 1.6, 3.1, and 0.8 microM, respectively. Fresh leaves of C. ambrosioides also contained isomeric hydroperoxides 6a and 7a, and the content ratio of 2a-7a suggested that these hydroperoxides were formed through the singlet-oxygen oxidation of limonene.
Assuntos
Chenopodiaceae/química , Peróxido de Hidrogênio/isolamento & purificação , Monoterpenos , Peróxidos , Plantas Medicinais/química , Terpenos/isolamento & purificação , Tripanossomicidas/isolamento & purificação , Trypanosoma cruzi/efeitos dos fármacos , Animais , Monoterpenos Cicloexânicos , Feminino , Células HeLa/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Japão , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sementes/química , Estereoisomerismo , Terpenos/química , Terpenos/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Neoplasias do Colo do ÚteroRESUMO
Trypanocidal constituents of dried leaves of Laurus nobilis L. (Lauraceae) were examined. Activity-guided fractionation of the methanol extract resulted in the isolation of two guaianolides, dehydrocostus lactone (1) and zaluzanin D (2), and a new p-menthane hydroperoxide, (1R,4S)-1-hydroperoxy-p-menth-2-en-8-ol acetate (3). The minimum lethal concentrations of these compounds against epimastigotes of Trypanosoma cruzi were 6.3, 2.5, and 1.4 microM, respectively.