Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV

Abstract

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI–BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.

Introduction

Shigella flexneri is a major cause of bacillary dysentery in developing countries (Bennish and Wojtyniak, 1991; Mikhail et al., 1990; Zaman et al., 1991) and is responsible for high morbidity and mortality, particularly in children under 5 years of age (Ferreccio et al., 1991; Kagalwalla et al., 1992). Shigella flexneri is divided into various serotypes based on the combination of antigenic determinants present in the O polysaccharide chains of the cell envelope lipopolysaccharide (LPS). This O polysaccharide is a polymer of tetrasaccharide repeating units: [-2)-α-l-RhaI-(1-2)-α-l-RhaII-(1-3)-α-l-RhaIII-(1-3)-β-d-GlcNAcp-(1-] shared by all serotypes except serotype 6 (Fig. 1). The basic tetrasaccharide polymer is the O-antigen of S. flexneri serotype Y and is designated as having group 3,4 antigen specificity (Kenne et al., 1978). Antigenic differences arise by the addition of a glucosyl and/or O-acetyl residue to a specific position on the basic tetrasaccharide repeating unit. Thus, strains expressing type V antigen have a glucosyl residue attached to the RhaII of the tetrasaccharide repeating unit (Fig. 1) (Kenne et al., 1977).

Genetic studies have revealed that the rfb (Macpherson et al., 1991) gene cluster and the rfc (Morona et al., 1994) gene coding for tetrasaccharide O-unit biosynthesis and polymerization, respectively, map adjacent to the his locus. Genes coding for O-acetylation and glucosylation, carried by temperate phages, are integrated near the pro-lac region on the S. flexneri chromosome (Petrovskaya and Licheva, 1982). Bacteriophage V (SfV) is responsible for type V antigen of S. flexneri. Shigella flexneri serotypes 5a and 5b are a consequence of the lysogenization of S. flexneri serotype Y and X by SfV, respectively.

Although a number of candidate S. flexneri vaccine strains have been constructed by various groups in the last 10 years, to date there is no vaccine available to control S. flexneri infections. SFL124 (ΔaroD), belonging to serotype Y, is an attenuated candidate vaccine strain (Lindberg et al., 1988, Lindberg et al., 1990) which has recently been shown to be safe and immunogenic in human volunteers (Li et al., 1993). It has been reported that natural S. flexneri infections in humans result in the production of an antibody response specific to the serotype of the infecting strain. Therefore it is likely that a vaccine must incorporate different serotype-specific antigens in order to induce an immune response against other serotypes of S. flexneri. This could be achieved by cloning the O-acetyl or glucosyl transferase (gtr) gene from temperate phages and then introducing them to SFL124 to create a hybrid vaccine strain which would have modified O-antigens on its surface. The O-acetyl and the gtr genes of phage Sf6 and SfX, respectively, have been cloned and characterized previously (Verma et al., 1991, Verma et al., 1993). In this paper we describe the cloning and sequencing of the bacteriophage SfV gene encoding glucosyl transferase, and the expression of the type V antigenic determinant in a serotype Y strain.