RESUMO
Theileria parva is a protozoan parasite transmitted by the brown ear tick Rhipicephalus appendiculatus that causes East Coast fever (ECF) in cattle, resulting in substantial economic losses in the regions of southern, eastern and central Africa. The schizont form of the parasite transforms the bovine host lymphocytes into actively proliferating cancer-like cells. However, how T. parva causes bovine host cells to proliferate and maintain a cancerous phenotype following infection is still poorly understood. On the other hand, current efforts to develop improved vaccines have identified only a few candidate antigens. In the present paper, we report the first comparative transcriptomic analysis throughout the course of T. parva infection. We observed that the development of sporoblast into sporozoite and then the establishment in the host cells as schizont is accompanied by a drastic increase of upregulated genes in the schizont stage of the parasite. In contrast, the ten highest gene expression values occurred in the arthropod vector stages. A comparative analysis showed that 2845 genes were upregulated in both sporozoite and schizont stages compared to the sporoblast. In addition, 647 were upregulated only in the sporozoite whereas 310 were only upregulated in the schizont. We detected low p67 expression in the schizont stage, an unexpected finding considering that p67 has been reported as a sporozoite stage-specific gene. In contrast, we found that transcription of p67 was 20 times higher in the sporoblast than in the sporozoite. Using the expression profiles of recently identified candidate vaccine antigens as a benchmark for selection for novel potential vaccine candidates, we identified three genes with expression similar to p67 and several other genes similar to Tp1-Tp10 schizont vaccine antigens. We propose that the antigenicity or chemotherapeutic potential of this panel of new candidate antigens be further investigated. Structural comparisons of the transcripts generated here with the existing gene models for the respective loci revealed indels. Our findings can be used to improve the structural annotation of the T. parva genome, and the identification of alternatively spliced transcripts.
Assuntos
Antígenos de Protozoários/genética , Perfilação da Expressão Gênica/métodos , Theileria parva/crescimento & desenvolvimento , Theileriose/parasitologia , Animais , Antígenos de Protozoários/imunologia , Bovinos , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Esquizontes/genética , Esquizontes/imunologia , Análise de Sequência de RNA/métodos , Esporozoítos/genética , Esporozoítos/imunologia , Theileria parva/genética , Theileria parva/imunologia , Regulação para CimaRESUMO
Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.
Assuntos
Linfócitos B/imunologia , Linfócitos/imunologia , Macrófagos/imunologia , Linfócitos T/imunologia , Theileria/imunologia , Theileriose/imunologia , Animais , Linfócitos B/parasitologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Citometria de Fluxo , Cavalos , Interações Hospedeiro-Parasita/imunologia , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Linfócitos/parasitologia , Macrófagos/parasitologia , Microscopia de Fluorescência , Parasitemia/imunologia , Parasitemia/parasitologia , Esquizontes/imunologia , Esquizontes/fisiologia , Imunodeficiência Combinada Severa/sangue , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/parasitologia , Especificidade da Espécie , Esporozoítos/imunologia , Esporozoítos/fisiologia , Linfócitos T/parasitologia , Theileria/fisiologia , Theileria annulata/imunologia , Theileria annulata/fisiologia , Theileria parva/imunologia , Theileria parva/fisiologia , Theileriose/parasitologiaRESUMO
OBJECTIVE: To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins. METHODS: Mice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum (P. falciparum) MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains. The positive clones were expanded to large (75 cm(2)) flask and cultured under the same conditions, checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible. RESULTS: A total number of 7 fusions including 26 plates (2 496 wells) were performed, of which 1 336 hybrids were produced and the overall efficiency (1 336/2496 × 100) was about 53%. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera. CONCLUSIONS: Bringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/química , Feminino , Humanos , Imunização , Malária/imunologia , Malária/parasitologia , Camundongos , Plasmodium falciparum/química , Plasmodium falciparum/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Esquizontes/crescimento & desenvolvimento , Esquizontes/imunologiaRESUMO
Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1ß, IL-6, IL-10, TNF, and transforming growth factor-ß), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.
Assuntos
Hemeproteínas/metabolismo , Malária/metabolismo , Plasmodium berghei/metabolismo , Plasmodium chabaudi/metabolismo , Pneumonia/parasitologia , Síndrome do Desconforto Respiratório/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Contagem de Linfócito CD4 , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Expressão Gênica , Hemeproteínas/fisiologia , Interações Hospedeiro-Parasita , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Malária/complicações , Malária/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Plasmodium berghei/imunologia , Plasmodium berghei/fisiologia , Plasmodium chabaudi/imunologia , Plasmodium chabaudi/fisiologia , Pneumonia/imunologia , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/etiologia , Esquizontes/imunologia , Esquizontes/metabolismo , Esquizontes/fisiologia , Especificidade da Espécie , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Murine cerebral malaria is a complex disease caused by Plasmodium berghei ANKA infection. Several cell types, including CD8(+) T cells, are essential effectors of disease. Although the use of transgenic parasites expressing model antigens has revealed the induction of cytotoxic T lymphocytes (CTL) specific for these model antigens, there is no direct evidence for a response to authentic blood-stage parasite antigens, nor any knowledge of its magnitude. Our studies show that there is a dramatic primary parasite-specific CTL response, akin to viral immunity, reaching approximately 30% of splenic CD8(+) T cells, with many producing interferon-γ and tumor necrosis factor-α. These cells express granzyme B and other markers of specific responders, are cytolytic, and respond to a broad array of major histocompatibility complex (MHC) I-restricted epitopes, 5 of which are identified here. Our studies indicate that vigorous CTL responses can be induced to pathogens even when they largely reside in red blood cells, which lack MHC I processing machinery.
Assuntos
Células Dendríticas/imunologia , Plasmodium berghei/imunologia , Esquizontes/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígeno CD11a/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Mapeamento de Epitopos , Epitopos/imunologia , Granzimas/metabolismo , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/crescimento & desenvolvimento , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/parasitologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Theileria parva is a tick-transmitted intracellular protozoan parasite that causes East Coast fever, a fatal bovine lymphoproliferative disease. The molecular mechanisms that underlie host cell transformation by T. parva schizonts have been studied extensively, and it is known that the nuclear factor-kappa B (NF-kappaB) is activated in schizont-infected cells, making T. parva-transformed cells resistant to apoptosis. However, the mechanism by which the parasite triggers the activation of NF-kappaB remains enigmatic. In the present study, we biochemically characterized a novel protein, which we termed TpSCOP (T. parvaschizont-derived cytoskeleton-binding protein), which is expressed in the schizont stage of T. parva. TpSCOP was shown to interact with F-actin in vitro. Expression of TpSCOP in a murine lymphocytic cell line resulted in the activation of NF-kappaB signaling pathways, leading to apoptosis resistance. The activation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was also detected. Furthermore, the introduction of TpSCOP into T. parva-infected cells also enhanced the activation of NF-kappaB. This is the first report to demonstrate that a parasite-derived molecule has the ability to activate the host NF-kappaB pathway. Based on these results, TpSCOP likely plays an important role in apoptosis inhibition during Theileria infection.
Assuntos
Linfócitos/imunologia , Linfócitos/parasitologia , Subunidade p50 de NF-kappa B/imunologia , Proteínas de Protozoários/imunologia , Esquizontes/imunologia , Theileria parva/imunologia , Actinas/metabolismo , Animais , Linhagem Celular , DNA de Protozoário/química , DNA de Protozoário/genética , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Camundongos , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Infection of erythrocytes with the Plasmodium parasite causes the pathologies associated with malaria, which result in at least one million deaths annually. The rupture of infected erythrocytes triggers an inflammatory response, which is induced by parasite-derived factors that still are not fully characterized. Induced secretion of inflammatory cytokines by these factors is considered a major cause of malaria pathogenesis. In particular, the inflammatory cytokine tumor necrosis factor (TNF) is thought to mediate most of the life-threatening pathologies of the disease. Here we describe the molecular characterization of a novel pathway that results in the secretion of TNF by host cells. We found that erythrocytes infected by Plasmodium accumulate high concentrations of hypoxanthine and xanthine. Degradation of Plasmodium-derived hypoxanthine/xanthine results in the formation of uric acid, which triggers the secretion of TNF. Since uric acid is considered a "danger signal" released by dying cells to alert the immune system, Plasmodium appears to have co-evolved to exploit this warning system. Identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.
Assuntos
Eritrócitos/parasitologia , Inflamação/parasitologia , Plasmodium/imunologia , Ácido Úrico/farmacologia , Animais , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Eritrócitos/metabolismo , Hipoxantina/metabolismo , Hipoxantina/farmacologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium yoelii/fisiologia , Esquizontes/efeitos dos fármacos , Esquizontes/imunologia , Esquizontes/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Xantina/metabolismoRESUMO
Research on malignant theileriosis is affected by the limited access to biological materials required for studies aiming at controlling the disease through the establishment of diagnostic tools and vaccines. The main aims of this work were to isolate, establish, and characterize a Theileria lestoquardi-infected cell culture (line) as a source of biological material and to generate a schizont cDNA library for further studies aiming at the identification of antigenic proteins. The T. lestoquardi isolate used originated from a sheep showing typical signs of malignant theileriosis in Atbara town in northern Sudan, and was maintained as an infected cell culture. A high-quality representative schizont cDNA library was established by isolating and purifying the schizonts using a nocodazole/aerolysin protocol followed by Percoll gradient ultracentrifugation. As a parameter to assess the quality of the schizont library, a provisional estimation of the percentage of recombinant phage clones originating from T. lestoquardi (Atbara) was undertaken. Ten clones with inserts ranging in size between 600 and 1200 bp were selected randomly, sequenced, and subjected to BLAST similarity searches. As 6 of the 10 sequenced clones showed similarities to T. parva, T. annulata, and other apicomplexan genes, it was concluded that the majority of the library phage clones originated from the parasite and not from host cell transcripts. The cDNA library will be used for screening of antigenic proteins using sera from infected sheep.