Glucosyl transferase gene(s) of temperate bacteriophages SfX and SfV and their role in the modification of the O-antigen of Shigella flexneri

Shigella flexneri is the main cause of bacillary dysentery in developing countries with a high attack rate and mortality in young children. Thus developing a S. flexneri vaccine has been given priority by WHO. It has been documented that Shigella lipopolysaccharide (LPS) is highly immunogenic. Antibodies directed against LPS play a major role in combating shigellosis, a protection that has been shown to be serotype specific. S. flexneri is divided into several serotypes, which differ in the structure of the O-antigen of the LPS. This O antigenic diversity is known to be phage dependent. The modification of S. flexneri O-antigen is due to genes encoding O-acetyl or glucosyl transferases of the temperate phage. Phages V (SfV) and SfX both carry a glucosyl transferase gene (gtr), which is responsible for the type V and the group specific antigen 7,8, respectively. We have cloned and sequenced the gtr genes of these two phages. The gtr gene product of the two phages share 35% homology. SFL124 serotype Y carrying the gtr of SfV or SfX was partially converted to serotypes 5a and X, respectively. The SfV genome was subsequently recloned to study the gene(s) required for full conversion. Genes located on the 5.0 kb BamHI fragment were found to be able to convert the S. flexneri serotype Y (SFL124) from serotype Y to 5a indistinguishably from 5a wild type. The 2.5 kb sequence of SfV contained the gtr gene and two other genes which conferred all genetic information for the glucosylation process. The full, and partial conversion was based on the relative level of the elimination of group antigen 3,4 as detected by immunoblotting, immunogold and the sensitivity of the sero converted strains to phage Sf6. The three genes formed a cluster located adjacent to the attP-int-xis region of SfV. The attP-int-xis of SfV was identified based on the high identity (80-90%) to that of the temperate P22 phage of Salmonella typhimurium. The X O antigenic conversion strain (SFL 1096) was immunogenic. Guinea-pigs immunized with 1x 108 c.f.u of S. flexneri SFL1096 were 75% protected from the attack of the homologous virulent strains. A panel of gtr genes of S. flexneri phages will be useful for developing a range of serotypes of live attenuated vaccine candidate strains of S. flexneri certain serotype.