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Infection and Immunity, August 2002, p. 4414-4423, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4414-4423.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health,¹ Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892,⁴ Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201,² Division of Virulence Assessment, Center for Food Safety and Nutrition, Food and Drug Administration, Laurel, Maryland 20708³

Received 26 December 2001/ Returned for modification 18 March 2002/ Accepted 8 May 2002

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.

Editor: J. D. Clements

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