Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

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.

Epitopes identified in GAPDH from Clostridium difficile recognized as common antigens with potential autoimmunizing properties

“…However, a novel approach was used to search for new immunoreactive proteins which is the use of umbilical cord blood sera. This method is routinely used in our laboratory 20 , 21 . It revealed a set of immunoreactive proteins (Fig.…”

“…Western blotting analysis was performed using umbilical cord blood sera from patients without the symptoms of CDI. The use of umbilical blood sera to identify new cross-reactive proteins with other pathogens have been practiced in our Laboratory for years 20 , 26 . As the transmission of antibodies from mother to the baby is highly regulated and selective process 27 , and therefore the transferred antibodies have extraordinary protective potential.…”

“…Our preliminary data show that sera from rabbits immunized with Lactobacillus johnsonii 142 38 does not specifically recognize P10 and P9 but other peptides from GAPDH sequence exhibit higher immunoreactivity (data not shown). It might be explained by that immune systems from different organisms elicit alternative antibody population to the same antigen 20 or that probiotic strains modulate immune response in such way that antibodies of different specificity are produced.…”

“…Selected peptides were synthesized using Geysen (PEPSCAN) procedure with changes 20 , 58 . Peptides were synthesized on polyethylene pins (MIMOTOPES, Clayton, Victoria, Australia) in a 96 well plate by adding one amino acid to each pin during one coupling reaction.…”

Layer-by-layer assembled membranes with immobilized porins

“…50 Previous studies have used this method to purify OmpF and OmpC, having functional proteins that were used in a lipid bilayer membrane and as antigen for the immune system, respectively. 51, 52 …”