RESUMEN
Adherence of Plasmodium falciparum-infected erythrocytes to the post-capillary endothelium is an important characteristic of malaria infection. The adhesion is mediated predominantly by P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a clonally variant protein expressed on the surface of infected red blood cells that appears to be a target of protective immunity. A multi-membered var gene family encodes PfEMP1 and switching expression of different var genes conveys different antigenic and adhesive properties to infected red blood cells. Knowledge about transcriptional control of phenotypic expression, or the mechanisms that allow multiple binding specificities, is very limited. Here, we describe a series of phenotypic selection experiments, which resulted in the expression of different PfEMP1 and the detection of multiple full-length var gene transcripts in the mature trophozoite stage. However, a dominant form of PfEMP1 appeared to be expressed, which suggested that most var transcripts do not lead to a surface expressed PfEMP1 molecule. Parasites bound to specific receptors still expressed multiple full-length var genes and mature trophozoites selected for increased adhesion to a specific receptor retained the ability to bind to multiple receptors. Our findings suggest that a defined adhesive phenotype can be associated with expression of multiple var genes.
Asunto(s)
Eritrocitos/inmunología , Eritrocitos/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Transcripción Genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Adhesión Celular , Cartilla de ADN , Endotelio Vascular/parasitología , Endotelio Vascular/fisiología , Endotelio Vascular/fisiopatología , Membrana Eritrocítica/inmunología , Membrana Eritrocítica/parasitología , Genes Protozoarios , Variación Genética , Humanos , Molécula 1 de Adhesión Intercelular/fisiología , Datos de Secuencia Molecular , Familia de Multigenes , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Mycoplasma pneumoniae/inmunología , Animales , Especificidad de Anticuerpos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Western Blotting , Cartilla de ADN/genética , Estudios de Evaluación como Asunto , Genes Bacterianos , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/genética , Peso Molecular , Mycoplasma pneumoniae/genética , Neumonía por Mycoplasma/diagnóstico , Neumonía por Mycoplasma/inmunología , Reacción en Cadena de la Polimerasa , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Pruebas Serológicas , Especificidad de la EspecieRESUMEN
Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (vlhA) from M. synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli. The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [3H]palmitate-labelled membrane proteins. Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family. The vlhA gene was shown to hybridize to multiple restriction fragments of the M. synoviae genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of M. synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.
