Abstract
Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections.
Highlights
The Shigella genus comprises of Gram-negative facultative human pathogens that are responsible for intestinal infections with symptoms including abdominal cramps, watery or bloody diarrhea and fever
This observation is in agreement with the earlier study by Roy et al [24], which suggested that the 35- to 38-kDa outer membrane proteins (OMP) are major antigens conferring a protective immune response in an animal model of shigellosis
Mice injected intraperitoneally with OmpC-monophosphoryl lipid A (MPL) mixture survived upon challenge with live bacteria, while mice immunized subcutaneously did not
Summary
The Shigella genus comprises of Gram-negative facultative human pathogens that are responsible for intestinal infections with symptoms including abdominal cramps, watery or bloody diarrhea and fever. In earlier studies we have described immunogenic and protective properties of the outer membrane proteins isolated from Shigella, Hafnia and E. coli strains [18,19] Both humoral [20] and cellular [21] protective responses were elicited after immunization of mice with these proteins. We have recently demonstrated [23] that outer membrane proteins from Shigella, Klebsiella, Hafnia and Citrobacter can react with human sera obtained from healthy individuals This observation is in agreement with the earlier study by Roy et al [24], which suggested that the 35- to 38-kDa OMPs are major antigens conferring a protective immune response in an animal model of shigellosis. We have identified a minimal amino acid sequence required for the antigenic activity of the peptide that represents the active epitope recognized by umbilical cord serum
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