Molecular cloning and characterization of genes for Shigella sonnei form I O polysaccharide: proposed biosynthetic pathway and stable expression in a live salmonella vaccine vector

Abstract

The gene region for biosynthesis of Shigella sonnei form I O polysaccharide (O-Ps) and flanking sequences, totaling >18 kb, was characterized by deletion analysis to define a minimal construct for development of Salmonella-based live vaccine vector strains. Lipopolysaccharide (LPS) expression and DNA sequence studies of plasmid deletion derivatives indicated form I O-Ps expression from a 12.3-kb region containing a putative promoter and 10 contiguous open reading frames (ORFs), one of which is the transposase of IS630. A detailed biosynthetic pathway, consistent with the predicted functions of eight of the nine essential ORFs and the form I O-Ps structure, is proposed. Further sequencing identified partial IS elements (i.e., IS91 and IS630) and wzz upstream of the form I coding region and a fragment of aqpZ and additional full or partial IS elements (i.e., IS629, IS91, and IS911) downstream of this region. The stability of plasmid-based form I O-Ps expression was greater from low-copy vectors than from high-copy vectors and was enhanced by deletion of the downstream IS91 from plasmid inserts. Both core-linked (i.e., LPS) and non-core-linked (i.e., capsule-like) surface expression of form I O-Ps were detected by Western blotting and silver staining of polyacrylamide gel electrophoresis-separated Shigella and Escherichia coli extracts. However, salmonellae, which have a core that is chemically dissimilar to that of shigellae, expressed only non-core-linked surface-associated form I O-Ps. Finally, attenuated Salmonella enterica serovar Typhi live vaccine vector candidates, containing minimal-sized form I operon constructs, elicited immune protection in mice against virulent S. sonnei challenge, thereby supporting the promise of live, oral vaccines for the prevention of shigellosis.

FIG. 1.

Cloning and downsizing of the S. sonnei form I biosynthetic gene cluster for sequencing and O-antigen expression studies. (A) Restriction map of the 30-kb BamHI insert from cosmid pXG914. (B) The inserts of plasmid subclones prepared to define a minimal essential region for form I O-antigen expression, defined by anti-form I-specific bacterial agglutination of recipient S. sonnei 53GII, E. coli HB101, or S. enterica serovar Typhi Ty21a carrying each of these plasmids. (C) Map of the form I gene region showing restriction sites relative to inserts shown in panel B and the location of 18 ORFs identified by sequence analysis. Filled ORFs represent the genes required for form I Ps biosynthesis in plasmid-bearing subclones. Restriction endonuclease sites are shown for BamHI (B), HindIII (H), PmeI (P), SmaI (S), and XbaI (X). (D) Percent G+C content of the 17,986-bp form I biosynthetic region and flanking sequences.

FIG. 2.

Detection of SDS-PAGE-separated O-Ps by silver staining and anti-form I Western immunoblotting with form I-specific antiserum. O-Ps is from S. sonnei 53GI or strain 53GII alone (control) or carrying plasmids with different form I-encoding inserts (A); E. coli HB101 alone (control) or carrying different form I-encoding plasmids (B); and S. enterica serovar Typhi Ty21a alone (control) or carrying different form I-encoding plasmids (C).

FIG. 3.

ORF diagrams of the regions flanking the S. sonnei form I biosynthetic gene cluster. (A) Regions of pWR101 and pWR102 upstream of wbgT. (B) Region of pWR101 downstream of wbgZ. The sequences of the left and right inverted repeats (IRL and IRR) of IS91 are shown in bold type. The gttc target sequence of IS91 is italicized. The original gttc sites within IS630 and IS911 for insertion of IS91 are boxed. A sequence homologous to a Pseudomonas IS element (accession number Y17830) occurs within the 263-bp hatched region.

FIG. 4.

Comparison of gene clusters for biosynthesis of the S. sonnei form I Ps and P. shigelloides O17 Ps. (A) Composite S. sonnei 53G form I gene cluster and flanking regions derived from sequences AF285971, AF294823, and AF455358. ORFs are identified numerically as defined in Table 2 and also by gene designations (38). (B) S. sonnei 53G form I gene cluster reported by Houng and Venkatesan (24). (C) Partial S. sonnei HW383 form I gene cluster determined by Chida et al. (6). (D) Composite P. shigelloides O17 Ps gene cluster derived from sequences AF285970 and AB025970. ORFs are identified numerically and by gene names (38). The ORFs associated with form I Ps biosynthesis are shaded.

FIG. 5.

Proposed pathway for biosynthesis of undecaprenyl phosphate (und-P)-linked, S. sonnei form I Ps. The pathway is based on the predicted enzymatic activities of S. sonnei 53G proteins as summarized in Table 2 and the structural steps required for conversion of UDP-GlcNAc to the putative form I Ps precursors, UDP-l-AltNAcA and UDP-4n-d-FucNAc.