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.
Xu
De-Qi
DQ
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cisar
John O
JO
Ambulos
Nicholas
N
Burr
Donald H
DH
Kopecko
Dennis J
DJ
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