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

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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 IS 630 . 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., IS 91 and IS 630 ) and wzz upstream of the form I coding region and a fragment of aqpZ and additional full or partial IS elements (i.e., IS 629 , IS 91 , and IS 911 ) 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 IS 91 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.