A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

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.

Development of a Multiplex PCR Assay Targeting O-Antigen Modification Genes for Molecular Serotyping of Shigella flexneri

“…flexneri is further divided into different serotypes based on the structure of the O antigen. At least 15 serotypes have been recognized: 1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, Xv, Y, and F6 (15,17,21). Serological analysis of S. flexneri has long been used to characterize isolates for epidemiological and bacteriological purposes.…”

“…The first two genes are highly homologous and interchangeable, whereas the third gene, gtr (type), is unique and encodes the serotype-specific glucosyltransferase (3,17). The serotype-specific gtr genes for types I, II, IV, and V, group 7;8, and 1c are gtrI, gtrII, gtrIV, gtrV, gtrX, and gtrIC, respectively (1,2,8,9,12,17). The gtr genes are encoded by different prophage genomes within the host chromosome.…”

“…The basic O antigen is referred to as serotype Y, and the addition of glucosyl and/or O-acetyl residues to the tetrasaccharide unit results in serological type (i.e., I, II, III, IV, and V)-, group (i.e., 3;4, 7;8, and 6)-, and 1c-specific antigenic determinants (17).…”

Serological Identification and Prevalence of a Novel O-Antigen Epitope Linked to 3- and 4- O -Acetylated Rhamnose III of Lipopolysaccharide in Shigella flexneri

“…Adding an O-acetyl group to Rha I at position 2 (2-O-acetylation) confers serotypes 1b, 3a, 3b, 4b, and 7b with group factor 6 (6,18,19). Glucosylation is mediated by three genes (gtrA, gtrB, and gtr [type specific]) that are arranged in a single operon known as the gtr cluster, which is encoded by serotype conversion bacteriophages, with 6 such bacteriophages identified to date (SfI, SfIC, SfII, SfIV, SfV, and SfX) (20)(21)(22)(23)(24)(25). 2-O-Acetylation of Rha I is performed by a specific O-acetyltransferase encoded by the gene oac (18,19), which is also carried by the serotype conversion bacteriophage Sf6 (26).…”

Serotype-Converting Bacteriophage SfII Encodes an Acyltransferase Protein That Mediates 6-O-Acetylation of GlcNAc in Shigella flexneri O-Antigens, Conferring on the Host a Novel O-Antigen Epitope

“…An acetyltransferase protein encoded by the oac gene which is carried on prophage Sf6 mediates the 2-Oacetylation (9,10). Glucosylation has been identified at different positions on one or two of the four sugar residues in the O-unit; it defines type I, IC, II, IV, and V as well as group 7,8 antigenic determinants in various serotypes (6,11). Three genes, gtrA, gtrB, and gtr (type specific), are responsible for the glucosylation in S. flexneri; they are carried on six prophages or cryptic prophages (SfI, SfIC, SfII, SfIV, SfV, and SfX) and are arranged in a single operon known as the gtr locus (10)(11)(12)(13)(14)(15)(16)(17).…”