RESUMEN

BACKGROUND: Thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis. METHODS: The 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays. RESULTS: Three members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro. CONCLUSIONS: We have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina.

Asunto(s)

Babesia/inmunología , Babesiosis/parasitología , Enfermedades de los Bovinos/parasitología , Merozoítos/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Trombospondina 1/química , Trombospondina 1/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Babesia/clasificación , Babesia/genética , Babesia/crecimiento & desarrollo , Bovinos , Femenino , Masculino , Merozoítos/química , Merozoítos/genética , Merozoítos/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Familia de Multigenes , Filogenia , Proteínas Protozoarias/genética , Alineación de Secuencia , Trombospondina 1/genética

RESUMEN

Eimeria zuernii and E. bovis are host-specific apicomplexan parasites of cattle causing haemorrhagic typhlocolitis in young animals worldwide. During first merogony, both Eimeria species form giant macromeronts (>300 µm) in host endothelial cells containing >120,000 merozoites I in vivo. During the massive intracellular replication of macromeronts, large amounts of cholesterol and fatty acids are indispensable for enormous merozoite I-derived membrane production. From a metabolic perspective, host endothelial cells might be of advantage to the parasite, as transcription of several genes involved in both, cholesterol de novo biosynthesis and low density lipoprotein (LDL)-mediated uptake, are up-regulated in Eimeria macromeront-carrying host endothelial cells. In order to analyse further influence of E. zuernii/E. bovis infections on the metabolism of cholesterol, fatty acids, and glycolysis of the host endothelial cells, suitable in vitro cell culture systems are necessary. So far, in vitro cell culture systems based on primary bovine umbilical vein endothelial cells (BUVEC) are available for E. bovis-macromeront I formation, but have not been evaluated for E. zuernii. A novel E. zuernii (strain A), initially isolated from naturally infected calves in Antioquia, Colombia, was used for sporozoite isolation. Primary BUVEC monolayers were concomitantly infected with E. zuernii- and E. bovis-sporozoites, resulting in large sized macromeronts whose morphological/morphometric characteristics were compared. BUVEC carrying E. zuernii-macromeronts resulted in the release of viable and highly motile merozoites I. Overall, E. zuernii-merozoites I differed morphologically from those of E. bovis. The new E. zuernii (strain A) will allow detailed in vitro investigations not only on the modulation of cellular cholesterol processing (i. e. cholesterol-25-hydroxylase and sterol O-acyltransferase) but also on the surface expression of LDL receptors during macromeront formation.

Asunto(s)

Enfermedades de los Bovinos/parasitología , Coccidiosis/veterinaria , Eimeria/crecimiento & desarrollo , Células Endoteliales/parasitología , Técnicas In Vitro/métodos , Animales , Bovinos , Coccidiosis/parasitología , Eimeria/citología , Eimeria/fisiología , Merozoítos/citología , Merozoítos/crecimiento & desarrollo , Merozoítos/fisiología , Esporozoítos/citología , Esporozoítos/crecimiento & desarrollo , Esporozoítos/fisiología

RESUMEN

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.

Asunto(s)

Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Plasmodium yoelii/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Eritrocitos/parasitología , Femenino , Humanos , Inmunización , Malaria/inmunología , Malaria/mortalidad , Malaria/parasitología , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/química , Vacunas contra la Malaria/genética , Masculino , Merozoítos/química , Merozoítos/crecimiento & desarrollo , Merozoítos/inmunología , Ratones , Ratones Endogámicos C57BL , Plasmodium yoelii/química , Plasmodium yoelii/genética , Plasmodium yoelii/crecimiento & desarrollo , Estructura Terciaria de Proteína , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Esporozoítos/química , Esporozoítos/crecimiento & desarrollo , Esporozoítos/inmunología

RESUMEN

Recently, we have described that apoptosis-like process of red blood cells (RBC) - eryptosis - in malaria is not restricted to parasitized cells, occurring also in non-parasitized RBC (nRBC). Besides to pathogenic proprieties, apoptosis also participates in the innate defense trough restriction of intracellular pathogens propagation. In the present study, we investigated the capacity of P. falciparum parasites to infect eryptotic RBC. Schizont parasitized RBC concentrated by magnetic separation were cultured with eryptotic RBC obtained by ionomycin treatment and, then, parasite growth was evaluated in Giemsa-stained thin blood smears. While parasites infected and developed normally in control non-eryptotic RBC, cultures performed with eryptotic RBC had a marked decrease in parasitaemia. It was noteworthy a great number of free merozoites in eryptotic RBC cultures, indicating that these cells were not susceptible to invasion. We suggest that although eryptosis could be involved in malaria pathogenesis, it could also acting protectively by controlling parasite propagation.

Asunto(s)

Apoptosis/fisiología , Eritrocitos/citología , Eritrocitos/parasitología , Malaria/patología , Parasitemia/patología , Plasmodium falciparum/patogenicidad , Animales , Humanos , Malaria/fisiopatología , Merozoítos/crecimiento & desarrollo , Parasitemia/fisiopatología

RESUMEN

Merozoite release from infected erythrocytes is a complex process, which is still not fully understood. Such process was characterised at ultra-structural level in this work by labelling erythrocyte membrane with a fluorescent lipid probe and subsequent photo-conversion into an electron-dense precipitate. A lipophilic DiIC(16) probe was inserted into the infected erythrocyte surface and the transport of this phospholipid analogue through the erythrocyte membrane was followed up during 48 h of the asexual erythrocyte cycle. The lipid probe was transferred from infected erythrocyte membranes to Maurer's clefts during merozoite release, thereby indicating that these membranes remained inside host cells after parasite release. Fluorescent structures were never observed inside infected erythrocytes preceding merozoite exit and merozoites released from infected erythrocyte were not fluorescent. However, specific precipitated material was localised bordering the parasitophorous vacuole membrane and tubovesicular membranes when labelled non-infected erythrocytes were invaded by merozoites. It was revealed that lipids were interchangeable from one membrane to another, passing from infected erythrocyte membrane to Maurer's clefts inside the erythrocyte ghost, even after merozoite release. Maurer's clefts became photo-converted following merozoite release, suggesting that these structures were in close contact with infected erythrocyte membrane during merozoite exit and possibly played some role in malarial parasite exit from the host cell.

Asunto(s)

Carbocianinas/metabolismo , Membrana Celular/metabolismo , Eritrocitos/parasitología , Merozoítos/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidad , Antígenos de Protozoos , Eritrocitos/química , Merozoítos/química , Plasmodium falciparum/química

RESUMEN

The Plasmodium falciparum ring-erythrocyte surface antigen (RESA)-like putative protein was identified and characterised. PCR and RT-PCR assays revealed that the gene encoding this protein was both present and being transcribed in P. falciparum strain FCB-2 16 h after erythrocyte invasion. Indirect immunofluorescence studies detected this protein in infected erythrocyte (IE) cytosol in dense fluorescent granules similar to Maurer's clefts at 16-20 h (parasites in ring and trophozoite stages) and very strongly on IE membranes at 22 h, suggesting that it is synthesised during early ring stages (16 h) and transported to the infected red blood cell (RBC) membrane surface during the trophozoite stage (22 h). Western blotting showed that antisera produced against polymerised synthetic peptides of this protein recognised a 72-kDa band in P. falciparum schizont lysate. P. falciparum RESA-like peptides used in normal RBC binding assays revealed that peptides 30326 ((101)NAEKI LGFDD KNILE ALDLFY(120)), 30334 ((281)RVTWK KLRTK MIKAL KKSLTY(300)) and 30342 ((431)SSPQR LKFTA GGGFC GKLRNY(450)) bind with high activity and saturability, presenting nM affinity constants. These peptides contain alpha-helical structural elements, as determined by circular dichroism, and inhibit P. falciparum in vitro invasion of normal RBCs by up to 91%, suggesting that some RESA-like protein regions are involved in intra-erythrocyte stage P. falciparum invasion.

Asunto(s)

Antígenos de Protozoos/metabolismo , Eritrocitos/metabolismo , Péptidos/metabolismo , Plasmodium falciparum/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Superficie/química , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Quimotripsina/metabolismo , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Cinética , Merozoítos/efectos de los fármacos , Merozoítos/crecimiento & desarrollo , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Unión Proteica , Transcripción Genética , Tripsina/metabolismo