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.
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.
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
De-Qi
DQ
Xu
John O
JO
Cisar
Nicholas
N
Ambulos
Donald H
DH
Burr
Dennis J
DJ
Kopecko
D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004195:Disease Models, Animal; D004405:Dysentery, Bacillary / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D019081:O Antigens / Q000096:biosynthesis / Q000235:genetics*; D012485:Salmonella typhi; D017422:Sequence Analysis, DNA; D022281:Shigella Vaccines / Q000235:genetics* / Q000276:immunology; D012764:Shigella sonnei / Q000235:genetics* / Q000276:immunology; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology
D004269:DNA, Bacterial; D019081:O Antigens; D022281:Shigella Vaccines; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic