ABSTRACT
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.
Subject(s)
Antibodies, Bacterial/biosynthesis , Bacterial Outer Membrane Proteins/analysis , Bottle Feeding , Breast Feeding , Escherichia coli Proteins/immunology , Escherichia coli Vaccines/immunology , Escherichia coli/pathogenicity , Feces/microbiology , Fimbriae Proteins/immunology , Antibodies, Bacterial/metabolism , Bacterial Outer Membrane Proteins/metabolism , Child, Preschool , Escherichia coli/immunology , Escherichia coli Infections/epidemiology , Escherichia coli Infections/immunology , Escherichia coli Infections/prevention & control , Escherichia coli Proteins/metabolism , Escherichia coli Vaccines/metabolism , Female , Fimbriae Proteins/metabolism , Humans , Infant , Infant, Newborn , Male , Prevalence , Vipoma/metabolismABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Vaccines/genetics , Escherichia coli Proteins/genetics , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Fimbriae Proteins/genetics , Animals , Humans , Immunoglobulin A/metabolism , Methylgalactosides/metabolism , Plasmids/metabolism , Rabbits , Recombinant Proteins/metabolism , Salmonella/metabolism , Thiogalactosides/metabolismABSTRACT
A plant expression cassette system was engineered to efficiently target proteins to the default secretory pathway, allowing the expression of DNA inserts in three frames as fusion proteins containing a synthetic tobacco calreticulin cleavable signal peptide sequence, with the advantage of producing the recombinant proteins by phytosecretion. As one approach to develop a vaccine to enteropathogenic Escherichia coli (EPEC) infection, the oral immunogenicity of phytosecreted BfpA, the structural subunit A of the bundle-forming pilus, expressed at high levels (7.7% of soluble protein) in transgenic tobacco tissues, was demonstrated in BALB/c mice by the induction and detection of fecal anti-BfpA antibodies.
