RESUMO
A diagnostic test was developed to discriminate active from past enteropathogenic Escherichia coli (EPEC) infection, which uses the affinity-purified recombinant proteins BfpA (bundle-forming pilus (BFP) structural repeating subunit A) and EspB (pore-forming secreted protein B) as reliable markers of virulence to detect antigen-specific coproantibodies by immunoblot analysis, and verification of active typical EPEC infection by gene-specific (bfpA and espB) PCR amplification using DNA extracted directly from specimens and/or culture-enriched preparations. To begin addressing the potential protective role of anti-EPEC antibodies at early age, the prevalence of IgA coproantibodies to these antigens was determined in either breastfed or artificially fed children <2 years of age hospitalized for watery diarrhea.
Assuntos
Anticorpos Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/análise , Alimentação com Mamadeira , Aleitamento Materno , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Escherichia coli/patogenicidade , Fezes/microbiologia , Proteínas de Fímbrias/imunologia , Anticorpos Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Pré-Escolar , Escherichia coli/imunologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/metabolismo , Vacinas contra Escherichia coli/metabolismo , Feminino , Proteínas de Fímbrias/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Vipoma/metabolismoRESUMO
BfpA, the structural repeating protein subunit A of the bundle-forming pilus and EspB, a type-III-secreted pore-forming protein of enteropathogenic Escherichia coli (EPEC), both virulence factors central for EPEC pathogenesis, were overexpressed in E. coli DH5alpha and M15 laboratory strains, respectively, using the pQE-30 cloning expression system, as chimeric fusions to a NH(2)-terminal histidine hexapeptide (His(6)-tag) sequence. After isopropyl beta-d-thiogalactoside induction, the expression levels achieved were 11 and 40% of total soluble protein for BfpA and EspB, respectively. The His(6)-tagged recombinant proteins were purified (up to 98% homogeneity) by Ni-agarose affinity chromatography and produced yields varying from 0.65 to 3.1 mg of recombinant protein per gram of wet weight cells. The immunogenicity and antigenicity of the final products were tested in rabbits and using fecal specimens obtained from children suffering from acute watery diarrhea, respectively. The recombinant products correspond to antigenically authentic protein standards, useful in future epidemiological and neonatal vaccinology studies.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Animais , Humanos , Imunoglobulina A/metabolismo , Metilgalactosídeos/metabolismo , Plasmídeos/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Salmonella/metabolismo , Tiogalactosídeos/metabolismoRESUMO
The frequency of IgA antibody activity to the structural protein subunit BfpA of the enteropathogenic Escherichia coli bundle-forming pilus was determined in 40 mother-infant pairs by immunoblot analysis using affinity purified recombinant BfpA to monitor for IgA in maternal colostrum and in feces of the neonates. Fecal samples were collected from exclusively breastfed term infants < 24-h after the first breastmilk feeding and colostral samples from their mothers. Infants were monitored prospectively with monthly visits to ascertain dietary practices and diarrheal illnesses. The percentage of colostral anti-BfpA IgA positive patients that were also coproantibody positive was 67.5%. The median duration of lactation was 108 days and the incidence of infantile diarrheal disease was 7.5%. Thus, colostral anti-BfpA IgA antibody activity survives passage through the gut of breastfed neonates, persisting in their feces. It is suggested that oral passive immunotherapy may be used to prevent and/or treat typical EPEC infection during infancy.
