RESUMO
Chagas disease is an important disease affecting millions of patients in the New World and is caused by a protozoan transmitted by haematophagous kissing bugs. It can be treated with drugs during the early acute phase; however, effective therapy against the chronic form of Chagas disease has yet to be discovered and developed. We herein tested the activity of solenopsin alkaloids extracted from two species of fire ants against the protozoan parasite Trypanosoma cruzi, the aetiologic agent of Chagas disease. Although IC50 determinations showed that solenopsins are more toxic to the parasite than benznidazole, the drug of choice for Chagas disease treatment, the ant alkaloids presented a lower selectivity index. As a result of exposure to the alkaloids, the parasites became swollen and rounded in shape, with hypertrophied contractile vacuoles and intense cytoplasmic vacuolization, possibly resulting in osmotic stress; no accumulation of multiple kinetoplasts and/or nuclei was detected. Overexpressing phosphatidylinositol 3-kinase-an enzyme essential for osmoregulation that is a known target of solenopsins in mammalian cells-did not prevent swelling and vacuolization, nor did it counteract the toxic effects of alkaloids on the parasites. Additional experimental results suggested that solenopsins induced a type of autophagic and programmed cell death in T. cruzi. Solenopsins also reduced the intracellular proliferation of T. cruzi amastigotes in infected macrophages in a concentration-dependent manner and demonstrated activity against Trypanosoma brucei rhodesiense bloodstream forms, which is another important aetiological kinetoplastid parasite. The results suggest the potential of solenopsins as novel natural drugs against neglected parasitic diseases caused by kinetoplastids.
Assuntos
Alcaloides/toxicidade , Venenos de Artrópodes/toxicidade , Tripanossomicidas/toxicidade , Trypanosoma cruzi/efeitos dos fármacos , Animais , Formigas/química , Apoptose , Autofagia , Células CHO , Cricetinae , Cricetulus , Macaca mulatta , Macrófagos/parasitologia , Pressão Osmótica , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A2 (PLA2), including Ca2+-independent iPLA2 and soluble, Ca2+-dependent sPLA2. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.
Assuntos
Leishmania , Pironas , Endocitose , Exocitose , NaftalenosRESUMO
Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and ß-tubulin. The major protein band recognized by host IgG was T. cruzi ß-tubulin. The T. cruzi ß-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi ß-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi ß-tubulin. A single immunization of mice with recombinant T. cruzi ß-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Imunoglobulina G/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Tubulina (Proteína)/imunologia , Animais , Modelos Animais de Doenças , Imunização , Masculino , Camundongos Endogâmicos BALB C , Camundongos MutantesRESUMO
All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.
Assuntos
Glicoproteínas/genética , Mucinas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/biossíntese , Complexo de Golgi/enzimologia , Estágios do Ciclo de Vida/genética , Mucinas/genética , Peptídeos/genética , Peptídeos/metabolismo , Polissacarídeos/biossíntese , Proteínas de Protozoários/genética , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
We investigated early cellular responses induced by infection with Leishmania major in macrophages from resistant C57/BL6 mice. Infection increased production of reactive oxygen species by resident, but not inflammatory peritoneal macrophages. In addition, infection increased activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) in resident, but not in inflammatory peritoneal macrophages. Infection also increased expression of membrane and soluble FasL, but infected macrophages remained viable after 48 h. Infection increased secretion of cytokines/chemokines TNF-α, IL-6, TIMP-1, IL-1RA, G-CSF, TREM, KC, MIP-1α, MIP-1ß, MCP-1, and MIP-2 in resident macrophages. Addition of antioxidants deferoxamine and N-acetylcysteine reduced ROS generation and JNK activation. Addition of antioxidants or JNK inhibitor SP600125 reduced secretion of KC. Furthermore, treatment with antioxidants or JNK inhibitor also reduced intracellular parasite replication. These results indicated that infection triggers a rapid cellular stress response in resident macrophages which induces proinflammatory signals, but is also involved in parasite survival and replication in host macrophages.
Assuntos
Leishmania major/fisiologia , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/parasitologia , Macrófagos/patologia , Macrófagos/parasitologia , Estresse Fisiológico , Animais , Antioxidantes/metabolismo , Morte Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Proteína Ligante Fas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Parasitos/efeitos dos fármacos , Parasitos/crescimento & desenvolvimento , Parasitos/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain.
Assuntos
Genoma de Protozoário , Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Acil Coenzima A/metabolismo , Acil Coenzima A/farmacologia , Clonagem Molecular , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Meios de Cultura , Ativação Enzimática , Ensaios Enzimáticos , Fumonisinas/farmacologia , Genes de Protozoários , Teste de Complementação Genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Filogenia , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Deleção de Sequência , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Tetra-Hidronaftalenos/farmacologia , Fatores de Tempo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus. METHODOLOGY/PRINCIPAL FINDINGS: Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H(+)-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs. CONCLUSIONS: We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.