Immunogenicity of the Shigella flexneri Serotype Y (SFL124) Vaccine Strain Expressing Cloned Glucosyl Transferase Gene of Converting Bacteriophage SfX

Abstract

The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O-antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild-type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O-antigen immunity in the host defense against shigellosis.

Type IV O antigen modification genes in the genome of Shigella flexneri NCTC 8296 The GenBank accession number for the sequence reported in this paper is AF288197.

“…Sensitivity to bacteriophage SfV and colony agglutination were used to determine if gtrA Ec gtrB Ec could completely complement gtrX. SFL1350 was resistant to infection by SfV (data not shown) whereas partially converted strain SFL1096 (SFL124 containing the gtrX gene only : Verma et al, 1993 ;Huan et al, 1995) was sensitive to SfV. These data were confirmed with colony agglutination where SFL1350 agglutinated strongly with group 7,8 sera compared to SFL1096, which reacted very weakly (data not shown).…”

“…The phenomenon of expressing dual serotypes, referred to as partial serotype conversion, on the surface of recombinant SFL124 has been observed previously (Adhikari et al, 1999 ;Guan et al, 1999 ;Huan et al, 1995Huan et al, , 1997a. When only the serotype-specific gtr gene is present, or when gtrA or gtrB are present along with the gtr gene, partial serotype conversion is observed and is proposed to result from the incomplete modification of the O antigen (Adhikari et al, 1999 ;Guan et al, 1999 ;Huan et al, 1995Huan et al, , 1997a.…”

“…When only the serotype-specific gtr gene is present, or when gtrA or gtrB are present along with the gtr gene, partial serotype conversion is observed and is proposed to result from the incomplete modification of the O antigen (Adhikari et al, 1999 ;Guan et al, 1999 ;Huan et al, 1995Huan et al, , 1997a. While the function of GtrA in O antigen modification is not known, it has been proposed to be a flippase that flips undecaprenol phosphate (UndP)-glucose from the inside of the cell to the outside of the cell where glucose is then transferred to the O antigen by the serotype-specific glucosyltransferase (Guan et al, 1999).…”

“…In the phage genome, the glucosylation genes are typically located downstream of the integrase gene and attP site, which mediates integration of the phage into the pro-lac region of the host genome (Adhikari et al, 1999 ;Guan & Verma, 1998 ;Petrovskaya & Licheva, 1982). The genes involved in O antigen modification are of interest because of their potential application in the development of S. flexneri vaccine strains expressing specific and, potentially, multiple serotypes (Guan & Verma, 1998 ;Huan et al, 1995 ;Verma et al, 1991).…”

The acid-resistance pathways of Shigella flexneri 2457T

“…The molecular weight marker SPP-1/EcoRI was used to determine DNA fragment sizes. Chromosomal DNA was prepared using the procedure described by Huan et al (1995).…”