Construction of recombinantaroAsalmonellae stably producing theShigella dysenteriaeserotype 1 O-antigen and structural characterization of theSalmonella/Shigellahybrid LPS

Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes

“…We have shown earlier that the introduction of plasmid pSS78 into E. coli K-12 or Salmonella typhimurium TV119, a rough Ra strain, generates a derivative expressing an LPS molecule containing one repeat unit of S. dysenteriae type 1 O antigen (4). In the present study, introduction of pSS78 into the rough strain S. flexneri SFL124-27 also generated a strain expressing only one repeat unit of S. dysenteriae type 1 O antigen ( Table 1).…”

“…Plasmid pSDM-4 is a pUC19 ampicillin-resistant hybrid plasmid carrying the rfp and rfb determinants of S. dysenteriae serotype 1 which specify biosynthesis of the O antigen of this organism (4,27). pMS26-2 was derived by insertion of the 4.8-kb SmaI fragment of pHP45 ⍀Hg (6) into the unique ScaI site located in the ampicillin resistance gene of pSDM-4, to create a mercury-resistant, non-ampicillin-resistant O antigen plasmid.…”

Production of glycoprotein vaccines in Escherichia coli

“…Recombinant EPA expressed from pGVXN150 contains two engineered N-glycosylation sites (N262 and N398) and is fused to a hexahistidine tag at the C-terminal end and to the DsbA signal peptide at the N-terminal end (Sec-dependent secretion to the periplasm). Tetracycline-selectable, low copy number plasmid pGVXN64 was used for biosynthesis of O antigen polysaccharides of S. dysenteriae serotype 1. pGVXN64 (origin of replication: IncPα) was constructed by insertion of an 11 kb Bam HI fragment of pSDM7 [18] containing the S. dysenteriae rfp and rfb gene clusters into the Bam HI site of pLAFR1 [19,20]. The rfp and rfb gene clusters encode glycosyltransferases and polymerases required for the synthesis of undecaprenyl-pyrophosphate-linked Shigella O1 polysaccharides [18] and were expressed from their native (constitutive) promoters in pGVXN64.…”

Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains

“…LPS was isolated as previously described (15) from S. dysenteriae 1 strain W30864 (wild type), E. coli K-12 DH5␣ harboring plasmid pSDM4 (rfp-rfb cassette [9]), and recombinant S. flexneri strains and subjected to electrophoresis on 15% polyacrylamide gels (polyacrylamide gel electrophoresis [24]). After Western blotting, the LPS was detected with a rabbit polyclonal antiserum against S. dysenteriae 1 LPS (Behringwerke, Marburg, Germany) as described previously (10).…”