RESUMEN
Wolbachia symbionts, first observed in the 1920s, are now known to be present in about 30-70% of tested arthropod species, in about half of tested filarial nematodes (including the majority of human filarial nematodes), and some plant-parasitic nematodes. In arthropods, they are generally viewed as parasites while in nematodes they appear to be mutualists although this demarcation is not absolute. Their presence in arthropods generally leads to reproductive anomalies, while in nematodes, they are generally required for worm development and reproduction. In mosquitos, Wolbachia inhibit RNA viral infections, leading to populational reductions in human RNA virus pathogens, whereas in filarial nematodes, their requirement for worm fertility and survival has been channeled into their use as drug targets for filariasis control. While much more research on these ubiquitous symbionts is needed, they are viewed as playing significant roles in biological processes, ranging from arthropod speciation to human health.
Asunto(s)
Artrópodos/microbiología , Filarioidea/microbiología , Simbiosis , Wolbachia , Animales , Interacciones Microbiota-HuespedRESUMEN
Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.
Asunto(s)
Marcadores Genéticos , Mansonella/genética , Mansoneliasis/diagnóstico , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN/métodos , África , Animales , Simulación por Computador , ADN Protozoario/genética , Pruebas Diagnósticas de Rutina , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mansonella/aislamiento & purificación , Técnicas de Diagnóstico Molecular , Enfermedades Desatendidas/diagnóstico , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , América del SurRESUMEN
The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.
Asunto(s)
Brugia Malayi/enzimología , Ciclosporina/química , Isomerasa de Peptidilprolil/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Brugia Malayi/genética , Cristalografía por Rayos X/métodos , Ciclosporina/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoAsunto(s)
Filarioidea/genética , Genes de Helminto , Inmunofilinas/genética , Proteínas de Unión a Tacrolimus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Brugia Malayi/química , Brugia Malayi/genética , Clonación Molecular , Dirofilaria immitis/química , Dirofilaria immitis/genética , Filarioidea/química , Inmunofilinas/antagonistas & inhibidores , Inmunofilinas/biosíntesis , Datos de Secuencia Molecular , Onchocerca volvulus/química , Onchocerca volvulus/genética , Proteínas Recombinantes/antagonistas & inhibidores , Alineación de Secuencia , Sirolimus/farmacología , Tacrolimus/farmacologíaAsunto(s)
Brugia Malayi/genética , Ciclosporina/farmacología , Mutagénesis , Isomerasa de Peptidilprolil/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Brugia Malayi/enzimología , Catálisis/efectos de los fármacos , Cartilla de ADN , Resistencia a Medicamentos/genética , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/efectos de los fármacos , Isomerasa de Peptidilprolil/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase activity and bind the immunosuppressive agent cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin-like domain was identified at the N-terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R-factor of 16.9% at 2.15 A resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.
Asunto(s)
Brugia Malayi/química , Isomerasa de Peptidilprolil/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Ciclosporina/metabolismo , Ciclosporina/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fragmentos de Péptidos/química , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/metabolismoAsunto(s)
Clonación Molecular , Dirofilaria immitis/enzimología , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Secuencia de Aminoácidos , Animales , ADN Complementario , Dirofilaria immitis/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad por SustratoAsunto(s)
Filarioidea/enzimología , Isomerasa de Peptidilprolil/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , ADN de Helmintos/química , Dirofilaria immitis/enzimología , Dirofilaria immitis/genética , Femenino , Filarioidea/clasificación , Filarioidea/genética , Datos de Secuencia Molecular , Peso Molecular , Isomerasa de Peptidilprolil/genética , Filogenia , ARN de Helminto/análisis , Alineación de SecuenciaAsunto(s)
Isomerasas de Aminoácido/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , Ciclosporina/farmacología , Filarioidea/enzimología , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/metabolismo , Secuencia de Aminoácidos , Animales , Brugia/enzimología , Brugia/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dirofilaria immitis/enzimología , Dirofilaria immitis/genética , Filarioidea/genética , Humanos , Datos de Secuencia Molecular , Onchocerca volvulus/enzimología , Onchocerca volvulus/genética , Isomerasa de Peptidilprolil , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Schistosoma/enzimología , Schistosoma/genética , Alineación de Secuencia , Toxoplasma/enzimología , Toxoplasma/genéticaRESUMEN
Dirofilaria immitis is an important filarial parasite of dogs and cats, and a useful model for human filariasis. Current diagnostic tests for heartworm infection in animals rely on the presence of fecund female worms (usually found 6.5 months post-infection or later) and therefore fail to detect pre-patent infections. Putative pepsin inhibitors from 2 filarial parasites of humans namely Onchocerca volvulus (Ov33, Oc3.6, OvD5B) and Brugia malayi (Bm33), have been shown to be useful in diagnosis of onchocerciasis and lymphatic filariasis, respectively. Previous studies have suggested that a homologue exists in D. immitis (DiT33), which may have potential in diagnosis of heartworm infection. In this study, the isolation and characterization of a cDNA clone encoding DiT33 is described. This cDNA contains 12 bases of the nematode-specific 22 nucleotide spliced leader sequence and encodes a 26.4 kDa-protein with a high level of similarity (87-89%) to other filarial members of the family. DiT33 was over-expressed in E. coli as a fusion with the maltose-binding protein and serological analysis was performed using a panel of clinically defined dog sera. The findings of this study indicate that DiT33 is a promising antigen for the early detection of D. immitis and may be a valuable tool in the control and management of heartworm infection.
Asunto(s)
Dirofilaria immitis/fisiología , Dirofilariasis/diagnóstico , Enfermedades de los Perros/diagnóstico , Filariasis/diagnóstico , Proteínas del Helminto/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , ADN Complementario , ADN de Helmintos/química , Dirofilariasis/inmunología , Perros , Femenino , Proteínas del Helminto/análisis , Proteínas del Helminto/química , Humanos , Datos de Secuencia Molecular , Onchocerca volvulus , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/química , Empalme del ARNRESUMEN
Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.
RESUMEN
The cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosporin A (CsA) to varying degrees. We have isolated a cDNA clone encoding a novel cyclophilin from the human filarial parasite Brugia malayi. This gene possesses an N-terminal domain homologous to cyclophilins from diverse phyla (49-60% amino acid sequence identity) and a hydrophilic C-terminal domain. The cyclophilin domain was overexpressed in Escherichia coli and found to possess peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 7.9 x 10(6) M-1 s-1. A histidine residue in lieu of tryptophan in the highly conserved CsA-binding site suggests that B. malayi cyclophilin is more closely related to the cyclophilin-like proteins described recently from natural killer (NK) cells, plants, and the 40 kDa cyclophilins from mammals. In accordance with the histidine-containing CsA-binding domain, the B. malayi enzyme was relatively insensitive to inhibition by CsA, since an IC50 value of 860 nM (compared to 19 nM for human cyclophilin A) was determined.
Asunto(s)
Isomerasas de Aminoácido/efectos de los fármacos , Brugia Malayi/genética , Proteínas Portadoras/efectos de los fármacos , Ciclosporina/farmacología , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Brugia Malayi/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In this study, the expression of an Onchocerca volvulus Ov33 homolog was demonstrated in Dirofilaria immitis. Rabbit antiserum raised against a recombinant fusion protein of O. volvulus, MBP/OvD 5B (Ov33), was found to react with a 31-33 kDa glycoprotein (DiT33) of adult worms of D. immitis. An antibody response to MBP/OvD 5B was observed in dogs, as early as 11 weeks post infection with infective larvae of D. immitis, and in dogs with occult infection. Cats both experimentally and naturally infected with D. immitis also reacted strongly with the recombinant antigen. In contrast, sera from dogs receiving chemically-abbreviated infection or from animals harbouring a variety of other helminths failed to react. These data suggest that antibody responses generated by DiT33 may have potential in immunodiagnosis of heartworm infection in cats and dogs.
Asunto(s)
Antígenos Helmínticos/inmunología , Dirofilaria immitis/inmunología , Dirofilariasis/diagnóstico , Onchocerca volvulus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/inmunología , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/inmunología , Gatos , Reacciones Cruzadas , Dirofilaria immitis/genética , Dirofilariasis/inmunología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Proteínas del Helminto/inmunología , Masculino , Datos de Secuencia Molecular , Onchocerca volvulus/genética , Conejos , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
In this study, dogs were immunized with irradiated L3 larvae of Dirofilaria immitis. Following challenge with non-irradiated L3, vaccinated dogs had an average of 71% fewer adult worms compared to non-vaccinated animals. A comparative analysis of eosinophil and antibody responses of these two groups of dogs is presented. Vaccinated dogs preferentially recognized several larval (14, 20, 30, 34, 39 kDa), adult worm (20 kDa) and microfilarial (36, 38, 71, 84 kDa) antigens. To characterize these antigens, the extent of glycosylation was assessed. The data suggest that an earlier response to these antigens may be important in the protection induced in dogs by administration of irradiated L3 of D. immitis.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Dirofilaria immitis/inmunología , Dirofilariasis/prevención & control , Enfermedades de los Perros/prevención & control , Vacunación/veterinaria , Animales , Antígenos Helmínticos/inmunología , Radioisótopos de Cobalto , Dirofilaria immitis/efectos de la radiación , Dirofilariasis/inmunología , Enfermedades de los Perros/inmunología , Perros , Eosinofilia/inmunología , Femenino , Glicoproteínas/análisis , Inmunización , Immunoblotting , Masculino , Proteínas Protozoarias/análisis , Vacunas Antiprotozoos/administración & dosificaciónAsunto(s)
Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo , Antígenos Helmínticos/análisis , Brugia/inmunología , Immunoblotting , Animales , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Antígenos de Superficie/análisis , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Brugia/genética , Reactivos de Enlaces Cruzados , Epítopos , Biblioteca Genómica , GlutaralRESUMEN
Anti-idiotypic (AB2) antibodies were generated in rabbits following immunization with a murine IgM monoclonal antibody (AB1) recognizing a surface determinant of Brugia malayi infective stage larvae. AB2 specifically inhibited the binding of AB1 to B. malayi larvae. Furthermore, AB2 had the ability to mimic the original antigen since mice immunized with AB2 possessed serum antibodies (AB3) specific for the B. malayi surface determinant. The presence of anti-surface antibodies (AB3 and AB1) induced either by AB2 immunization or by administration of AB1, did not alter the outcome of an intraperitoneal infection of B. malayi larvae in BABL/c mice when compared to untreated animals. AB3 antibodies like AB1, were IgM, thus indicating an isotype restricted response to the B. malayi epitope. There were no detectable cell mediated responses to the surface determinant in mice immunized with AB2, assessed by lymphocyte blastogenesis or IL3 production in vitro in response to the idiotope as presented by living larvae. The lack of cellular responses and/or the previously demonstrated rapid shedding of the epitope may explain the inability of AB1 or AB2 to protect mice against larval challenge in this study.
Asunto(s)
Anticuerpos Antiidiotipos , Anticuerpos Antihelmínticos , Antígenos Helmínticos , Brugia/inmunología , Animales , Anticuerpos Antihelmínticos/biosíntesis , Antígenos de Superficie , Filariasis Linfática/prevención & control , Epítopos , Femenino , Isotipos de Inmunoglobulinas/biosíntesis , Larva/inmunología , Ratones , Ratones Endogámicos BALB C , Vacunas/inmunologíaRESUMEN
We analyzed the antigenicity and stability of the surface of skin microfilariae (mf) of Onchocerca cervicalis, a horse parasite. These mf express antigens on their surface that are cross-reactive with the cattle parasite O. lienalis and with the human parasite O. volvulus. The surface of living O. cervicalis mf was radioiodinated using Iodogen and the labeled components were solubilized in buffers containing sodium dodecyl sulphate (SDS), or extracted with the milder detergent octyl-beta-D-glucopyranoside (OGP). Electrophoresis of this material showed seven prominent bands, one of which (14 kDa) was specifically precipitated by antisera from rabbits immunized with mf from either O. cervicalis, O. lienalis, or O. volvulus, and by human sera obtained from infected individuals in Chiapas, Mexico. Other components were precipitated by either the rabbit or the human sera. In addition, antisera from mice immunized with O. cervicalis mf bound specifically to the surface of freeze-thawed uterine O. lienalis and O. volvulus mf as detected by immunofluorescence. This fluorescence was lost from the surface of O. cervicalis mf in a temperature-dependent fashion. Live mf incubated on ice with mouse anti-mf antisera and secondary FITC-GAM, showed uniform surface fluorescence. When these mf were incubated at 37 degrees C, but not at 0 degrees C, the fluorescent pattern changed with time. First, small non-fluorescent patches arose, followed by an increasingly wide belt devoid of fluorescence, and finally, no visible fluorescence. These changes in the mf surface suggest potential mechanisms for immune evasion by filarial parasites.
Asunto(s)
Antígenos Helmínticos/análisis , Onchocerca/inmunología , Animales , Antígenos de Superficie/análisis , Autorradiografía , Western Blotting , Bovinos , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Caballos , Humanos , Inmunidad Celular , Pruebas de Precipitina , Piel/parasitología , Propiedades de SuperficieRESUMEN
The model of Onchocerca lienalis microfilariae (mf) in CBA mice has been employed to examine the immunological mechanisms underlying the destruction of skin-dwelling mf in onchocerciasis. Comparative studies among immunologically intact (CBA/H) or deficient (CBA/N, T-cell-deprived) syngeneic animals demonstrated that levels of mf of a primary infection were reduced most rapidly in fully immunocompetent mice. Significant reductions in recoveries of a secondary infection were evident in CBA/H (80%) and CBA/N (44%) mice, but not in T-cell-deprived animals. The establishment of primary and secondary infections was apparently not influenced by complement, as judged by C3 depletion with Cobra Venom Factor. Eosinophilia was demonstrated to varying degrees in all infected animals; similar levels occurred in CBA/H and CBA/N mice which were greatly elevated after mf challenge. In contrast, the eosinophil response of T-cell-deprived mice was weak and not potentiated during secondary infection. Type I immediate hypersensitivity responses to soluble mf antigen (mf-Ag) were mounted by all groups, but significantly less strongly in T-cell-deprived mice. Type IV delayed responses were generally weak, although CBA/N mice reacted strongly in the early phase of primary infections. During the first 2 weeks of infection CBA/H and T-cell-deprived mice mounted rapid IgM responses to mf-Ag. Subsequently, levels of IgG2a, IgG2b and IgG1 increased in all mice. There was a potentiated IgG2a, IgG2b and IgG1 response in all groups following challenge, with levels of IgG1 highest in CBA/H mice. IgE responses were also detected by passive cutaneous anaphylaxis during primary and secondary infections. Peak levels of parasite-specific antibodies coincided with the timing of mf clearance.