RESUMO
The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced. All derivatives but one (SL3235) stably inherited the new trait. Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS. Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccharide with the following structure, [formula: see text] demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core.
Assuntos
Alquil e Aril Transferases , Proteínas de Bactérias/genética , Lipopolissacarídeos/biossíntese , Proteínas Nucleares/genética , Antígenos O/biossíntese , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Shigella dysenteriae/genética , Transferases/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase , Proteínas de Bactérias/metabolismo , Bacteriófago P22/genética , Configuração de Carboidratos , Sequência de Carboidratos , Elementos de DNA Transponíveis/genética , Lipídeo A/biossíntese , Lipídeo A/química , Lipopolissacarídeos/química , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Antígenos O/imunologia , Salmonella/genética , Salmonella/metabolismo , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Shigella dysenteriae/imunologiaRESUMO
The potential utility of Shigella flexneri aroD vaccine candidates for the development of bi- or multivalent vaccines has been explored by the introduction of the genetic determinants rfp and rfb for heterologous O antigen polysaccharide from Shigella dysenteriae serotype 1. The serotype Y vaccine strain SFL124 expressed the heterologous antigen qualitatively and quantitatively well, qualitatively in the sense of the O antigen polysaccharide being correctly linked to the S. flexneri lipopolysaccharide R3 core oligosaccharide and quantitatively in the sense that typical yields were obtained, with ratios of homologous to heterologous O antigen being 4:1 for one construct and 1:1 for another. Moreover, both polysaccharide chains were shown to be linked to position O-4 of the subterminal D-glucose residue of the R3 core. In contrast to the hybrid serotype Y SFL124 derivatives, analogous derivatives of serotype 2a vaccine strain SFL1070 did not elaborate a complete heterologous O antigen. Such derivatives, and analogous derivatives of rough, O antigen-negative mutants of SFL1070, formed instead a hybrid lipopolysaccharide molecule consisting of the S. flexneri lipid A R3 core with a single repeat unit of the S. dysenteriae type 1 O antigen. Introduction of the determinants for the S. dysenteriae type 1 O antigen into a second serotype 2a strain and into strains representing other serotypes of S. flexneri, revealed the following for the expression of the heterologous O antigen: serotypes 1a, 1b, 2a, and 5a did not produce the heterologous O antigen, whereas serotypes 2b, 3a, 3b, 4a, 4b, 5b, and X did.
Assuntos
Polissacarídeos Bacterianos/biossíntese , Shigella dysenteriae/imunologia , Shigella flexneri/imunologia , Proteínas de Bactérias/genética , Vacinas Bacterianas , Configuração de Carboidratos , Sequência de Carboidratos , Genes Bacterianos , Metilação , Dados de Sequência Molecular , Proteínas Nucleares/genética , Antígenos O , Oligossacarídeos/química , Polissacarídeos Bacterianos/química , Sorotipagem , Shigella dysenteriae/genética , Shigella flexneri/classificação , Shigella flexneri/genética , Vacinas Atenuadas , Vacinas SintéticasRESUMO
Mouse and rat monoclonal antibodies were generated against lipopolysaccharides expressed in Escherichia coli K-12/Shigella dysenteriae type 1 hybrids and native Shigella dysenteriae type 1 bacteria. Six monoclonal antibodies, all IgM and reacting with the E. coli K-12/Shigella dysenteriae type 1 hybrids, were selected and characterized. The specificities were studied in inhibition experiments using purified native and synthetic saccharides, representing different parts of the S. dysenteriae type 1 tetrasaccharide repeating unit. Three patterns of specificities were seen: (i) MAEC-SD-1 and MAEC-SD-2 recognizing the terminal alpha-D-Galp-(1-->3)-beta-D-GlcpNAc disaccharide as present in an E. coli K-12 class I hybrid strain, (ii) MAEC-SD-11; a class IV specific mAb, recognizing the alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->tris accharide, and (iii) MAEC-SD-21, MASD-1, MASD-2; all mAbs with class V specificity, recognizing the alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1--> trisaccharide. Furthermore, the data strongly suggest that the native repeating unit for S. dysenteriae type 1 is the alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1--> 3)-alpha-D-GlcpNAc tetrasaccharide.
Assuntos
Anticorpos Antibacterianos/imunologia , Epitopos/imunologia , Oligossacarídeos/imunologia , Polissacarídeos Bacterianos/imunologia , Shigella dysenteriae/imunologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Sequência de Carboidratos , Cruzamentos Genéticos , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Antígenos O , Polissacarídeos Bacterianos/genética , Shigella dysenteriae/genéticaRESUMO
The structures of the polysaccharide part of lipopolysaccharides isolated from eight Escherichia coli K12/Shigella dysenteriae type 1 hybrids have been determined using sugar and methylation analysis plus 1H- and 13C-nuclear magnetic resonance spectroscopy. The hybrids express parts of the S. dysenteriae type 1 O-antigen tetrasaccharide repeating unit because of the presence of pSS3, a plasmid expressing an alpha-galactosyl: lipopolysaccharide transferase and pSS9, a pBR322 plasmid expressing S. dysenteriae type 1 rfb genes. The various classes of hybrids are the result of transposon Tn 1000 insertions in pSS9 inactivating different rfb genes. The following structural elements were found. E. coli K12 (pSS3) and E. coli K12 (pSS3, pSS9-6; a class I hybrid); alpha-D-Galp(1-->3)beta-D-GlcpNAc(1-->. Class IV hybrids: E. coli K12 (pSS3, pSS9-36); (pSS3, pSS9-107) and (pSS3, pSS9-114); alpha-L-Rhap(1-->2)alpha-D-Galp(1-->3)beta-D-GlcpNAc(1-->. Class V hybrids: E. coli K12 (pSS3, pSS9-78) and (pSS3, pSS9-111); alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)alpha-D-Galp(1-->3)bet a-D-GlcpNAc(1-->. The structural sequences are identical to those found in the lipopolysaccharide from native S. dysenteriae type 1. In the hybrid strains, the terminal non-reducing GlcNAc residue of the E. coli K12 core is fully substituted by S. dysenteriae type 1 repeating units, or parts thereof.
