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    ABSTRACT: Glucosyltransferases (Gtrs) and O-acetyltransferase (Oac) are integral membrane proteins embedded within the cytoplasmic membrane of Shigella flexneri. Gtrs and Oac are responsible for unidirectional host serotype conversion by altering the epitopic properties of the bacterial surface lipopolysaccharide (LPS) O-antigen. In this study, we present the membrane topology of a recently recognized Gtr, GtrIc, which is known to mediate S. flenxeri serotype switching from 1a to 1c. The GtrIc topology is shown to deviate from those typically seen in S. flexneri Gtrs. GtrIc has 11 hydrophilic loops, 10 transmembrane helices, a double intramembrane dipping loop 5, and a cytoplasmic N- and C-terminus. Along with a unique membrane topology, the identification of non-critical Gtr-conserved peptide motifs within large periplasmic loops (N-terminal D/ExD/E and C-terminal KK), which have previously been proven essential for the activity of other Gtrs, challenge current opinions of a similar mechanism for enzyme function between members of the S. flexneri Gtr family.
    Molecular Membrane Biology 04/2010; 27(2-3):114-22. · 3.13 Impact Factor
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    ABSTRACT: The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.
    Journal of bacteriology 09/2009; 191(21):6612-7. · 3.94 Impact Factor
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    ABSTRACT: Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.
    Microbiology and Immunology 02/2007; 51(11):1045-52. · 1.55 Impact Factor
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    ABSTRACT: The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.
    Clinical and Diagnostic Laboratory Immunology 12/2003; 10(6):1078-84. · 2.51 Impact Factor

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