Siegfried Schlecht

IDENTIFICATION OF A NOVEL CORE TYPE IN SALMONELLA LIPOPOLYSACCHARIDE : COMPLETE STRUCTURAL ANALYSIS OF THE CORE REGION OF THE LIPOPOLYSACCHARIDE FROM SALMONELLA ENTERICA SV. ARIZONAE O62

 For the first time, the complete structure of a lipopolysaccharide (LPS) core region fromSalmonella enterica has been identified that is different from the Ra core type generally thought to be present in allSalmonella LPS. The LPSs from two rough mutants and the smooth form of S. enterica sv. Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy. In the novel core type, the terminal d-GlcNAc residue present in the Ra core type, is replaced by a d-Glc residue. The O-specific polysaccharide is α1→4-linked to the second distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 ofl-glycero-d-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II). R α 1 → 4 Glc 2 ↑ Glc p α 1 p α 1 → Gal p α 1 → 3 Glc 6 ↑ Gal p α 1 p α 1 P ↓ 4 → 3 Hep 7 ↑ Hep p α 1 p α 1 → 3 Hep PPE ↓   4 p α 1 A → 5 Kd 4 ↑ Kdo α 2 o STRUCTURE I Abbreviations in Structure I are as follows: Hepp,l-glycero-d-manno-heptopyranose; Kdo, 3-deoxy-d-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide. The presence of this novel core type in LPS of S. entericashould be taken into account in the development of a general antibody-based diagnostic system forSalmonella.

A murine monoclonal antibody that recognizes a genus-specific epitope in the Salmonella lipopolysaccharide outer core.

A murine monoclonal antibody 105 made from spleen cells of a mouse immunized with a mixture of common Salmonella serotypes reacted specifically with salmonellae from the most frequently encountered O serogroups of A (O:2) to E (O:3), and with strains from the less common O serogroups that represent the subspecies I, II, IIIb, IV, V and VI. Specificity for Salmonella was demonstrated by the lack of reactivity of monoclonal antibody 105 with any of the 30 other different species of Gram-positive and Gram-negative bacteria tested including 16 species in the family of Enterobacteriaceae. Studies to elucidate its binding epitope have shown that it reacts with the three distal sugar residues joined through specific anomeric linkages as present only in the Salmonella lipopolysaccharide outer core, which explains its specificity for the Salmonella. The failure of monoclonal antibody 105 to react with a subspecies IIIa Salmonella suggested a different outer core structure in this strain of Salmonella and also that monoclonal antibodies to the outer core of Salmonella lipopolysaccharide should be useful in the molecular analysis of their diversity.

Lack of the α-1,2-linked N-acetyl-D-glucosamine epitope in the outer core structures of lipopolysaccharides from certain O serogroups and subspecies of Salmonella enterica

A total of 176 strains of Salmonella enterica representing 116 serotypes were tested for the presence of the T6 epitope of the alpha-1,2-linked N-acetyl-D-glucosamine residue by reaction with a murine monoclonal antibody T6 specific for this structure in the Salmonella Ra core lipopolysaccharide (LPS). All 20 serotypes (70 strains) belonging to serogroups A to E were positive for the T6 epitope while 29% of the 96 serotypes (106 strains) belonging to O serogroups F to 67 were negative; 12 serotypes (12 strains) of subspecies IIIb Salmonella were positive for the T6 epitope, but 10 serotypes (11 strains) of subspecies IIIa Salmonella were found to lack this epitope. In T6-positive strains, the epitope was accessible to antibody binding in both the unsubstituted free rough core LPS and in the rough core LPS substituted with a few repeating units of O side chains. The presence or absence of the T6 epitope in Salmonella strains was not affected by culture conditions, the source of the isolate, the age of the culture or the presence of fimbriae antigens.

Enhancement of Protection against Salmonella Infection in Mice Mediated by a Synthetic Lipopeptide Analogue of Bacterial Lipoprotein in S. typhimurium Vaccines

Vaccines consisting of acetone-killed Salmonella typhimurium were supplemented with a synthetically prepared lipopeptide derivative of bacterial lipoprotein, Pam3Cys-Ser-Ser-Asn-Ala. NMRI mice were immunized with these vaccines, receiving two intraperitoneal injections and were challenged intraperitoneally with graded doses of S. typhimurium C5. The protective capacity of the supplemented vaccines was compared with that of the unsupplemented bacterial vaccine, and with the effectiveness of the supplementing component alone. The LD50 served as a criterion for protective capacity. The results showed that 90% of the S. typhimurium S-form vaccine could be replaced by the adjuvant lipopeptide without a recognizable decrease in protective immunizing capacity. A similar but less pronounced enhancement of protection was obtained with a R-mutant vaccine supplemented with the lipopeptide; by supplementing the standard vaccine dose with lipopeptide an increase in protection was also achieved. Lipopeptide alone was not effective in protecting mice from infection with S. typhimurium.

Lipopeptide als natürliche Adjuvantien für Impfstoffe aus Gram-negativen Bakterien

Bacterial cell wall components such as lipopolysaccharide, a variety of membrane proteins, murein, and lipoprotein can act as immunoadjuvants for bacterial vaccines, thus enhancing protection from bacterial infections. Synthetically prepared N-terminal parts of the lipoprotein from Enterobacteria carrying three fatty acid residues or lipopeptide analogs containing one to four aminoacids bound to S-glycerylcysteine act as potent immunoadjuvants in vivo in combination with or covalently linked to antiges. Here we demonstrate that the supplementation ofSalmonella vaccines with these synthetic lipopeptides significantly enhances their vaccine efficiency in mice. Variations in the native lipopeptide structure regarding chain length and amino acid sequence of the peptide moiety, as well as modifications of the lipoamino acid, lead to reduction or even complete loss of the adjuvant activity. The immunoadjuvant properties of the lipopeptides as described here are mediated by an enhancement of the humoral immune response.

Structural differences in the outer core region of lipopolysaccharides derived from members of the genus Salmonella

Spontaneous and P22-resistant rough mutants, respectively, selected from Salmonella IV (18: z36, z38:-) and S. djakarta (48: z4, z24:-), appeared to lack the epitope recognized by the T6 monoclonal antibody which had been previously shown to correspond to the terminal alpha-1,2-linked N-acetyl-D-glucosamine residue of the Salmonella lipopolysaccharide (LPS) Ra core. LPSs and core oligosaccharides were therefore prepared from these two rough mutants and analysed by chemical and serological methods. Sugar analyses as well as methylation and 13C-NMR studies indicated that rough mutants derived from these two serotypes indeed possessed outer core structures differing from those of the well-characterized Salmonella Ra core. Serological data corroborated the chemical findings. Proposed structures of the outer core regions of these two R-types are presented and the significance of the findings is discussed.

Extraction and biochemical analyses ofHelicobacter pylori lipopolysaccharides

Lipopolysaccharides were isolated from dehydratedHelicobacter pylori cells by the phenol-chloroform-petroleum ether and hot phenol/water extraction techniques. Biochemical characterization of the crude extracts indicated the following: The primary fatty acids and approximate molar ratios were 3-hydroxyoctadecanoic (2), 3-hydroxyhexadecanoic (1), and octadecanoic (1) acids. Lesser amounts of tetradecanoic, hexadecanoic, and octadecenoic acids were noted. 3-Hydroxytetradecanoic acid was not deteted in either extract. Neutral sugar analyses detected glucose, galactose, two heptose isomers, and an unidentified deoxy-hexose. Glucosamine and glucosamine phosphate were the only amino sugars found in significant quantities. Analyses of other components included ethanolamine, phosphate, and protein. 3-Deoxy-d-manno-octulosonic acid (KDO) was detected, but in lower concentrations than would be expected in comparable enterobacterial lipopolysaccharides.

Molecular organization of the outer membrane of Salmonella typhimurium. Different release of lipopolysaccharide from wild type and lipopolysaccharide mutant cells by EDTA treatment.

Cells of Salmonella typhimurium wild type and of several well defined lipopolysaccharide mutants were treated with EDTA. The percentage release of lipopolysaccharide and phospholipid was determined. The results obtained show that the release of lipopolysaccharide by EDTA declines along with the gradually diminishing chain length of the lipopolysaccharide, althought the total amount of lipopolysaccharide was found to increase at the same time in the respective mutants. Implications of these findings for the organization of the outer membrane are discussed.

Immunoblot Analysis of the R-form Lipopolysaccharide from Salmonella S forms

Lipopolysaccharide (LPS) preparations from Salmonella S-form bacteria contain, in addition to S-form LPS, variable amounts of R-form material, lacking the O-specific polysaccharide. In the present study, we investigated the R-form LPS present in 22 LPS preparations derived from different Salmonella S-form strains belonging to 8 serological groups. The R-form part of the LPSs was separated by SDS-polyacrylamide gel electrophoresis and its antigen specificity analyzed by subsequent immunoblotting using Salmonella R-antisera (anti-Ra, -Rb1, -Rb2, -Rc). The results show that different R-determinants were present in one and the same LPS preparation. The pattern of reaction varied considerably among individual strains. This variation was also present among strains of the same serogroup. Approximately one half of the R-form LPSs exhibited a weak or no reaction with Ra antiserum. In contrast, almost all R-form LPSs showed a significant reaction with Rb1 antiserum. The electrophoretic mobility of the R-form material was very similar, exhibiting only minor differences among the different LPS preparations. In most cases, migration was similar to that of authentic Salmonella Ra-LPS; in some cases, the migration was somewhat faster resembling that of Rb1-LPS.

Schutzversuche gegen S. typhimurium-Infektion an Mäusen. Chemische Zusammensetzung und biologische Wirksamkeit verschiedener Extrakte aus Salmonella-R-Mutanten

Abstract It is not clear as yet how far defined structural components in gram-negative bacteria participate in the induction of immunity to (gram-negative) infections. A number of bacterial components have been considered as playing a role in this respect. These components include the O-antigenic determinants and the more ubiquitous structures of the core portion (Ra-Re) of lipopolysaccharides. Also proteins have been discussed in this connection, especially surface proteins that are exposed under in vivo conditions. In addition, even internal constituents such as nucleic acids have been thought to play a role in induction of immunity to infection. Since it is not yet clear whether all of these components are necessary, we have reinvestigated this question. In the present communication we compare a number of bacterial extracts with respect to their chemical composition and their ability to protect mice against infection to S. typhimurium . Bacteria from a Salmonella typhimurium S form and from 5 R-mutant strains from S. typhimurium and S. minnesota were subjected to extraction with guanidine thiocyanate, urea and veronal buffer. Subsequent analysis revealed significant differences with respect to yield and composition of the antigenic material thereby obtained. With live bacteria, highest yields were obtained by extraction with guanidine thiocyanate (13–23% of the bacterial dry masses) and lowest with veronal buffer (1–3%), while yields obtained by urea were between these two. In the latter case prior killing of the cells with phenol significantly increased the amount of material obtained. One essential difference among the isolation procedures lay in their efficiency for the quantitative extraction of cell wall proteins, which constituted the major part of all extracts. In contrast, with all the procedures the amounts of lipopolysaccharide, phospholipid and nucleic acid obtained were comparable and were relatively low. The highest protein (about 90% of extracted material) and thereby correspondingly lowest LPS- and phospholipid content were exhibited by the guanidin thiocyanate extracts. The lowest protein-(60–80%) and highest LPS and phospholipid content however, were found in the veronal extracts. SDS-polyacrylamide gel electrophoresis of the guanidine thiocyanate and urea extracts from the same bacterial strains showed the presence of similar polypeptides, however in a different quantitative distribution. In all extracts obtained by the two methods 18 common polypeptides were present. The veronal buffer extracts of the mutants on the other hand showed a lower number of polypeptides of which only 5–6 were common. None of the above methods led to the isolation of significant amounts of proteins of porin complex origin. In the individual extracts up to 30 polypeptides were detectable. In active immunization experiments using NMRI mice it could be shown that the extracts afford protection against infection with S. typbimurium . Protective activity was measured as the LD 50 in immunized animals. All extracts from a S. typbimurium Ra mutant (and also those from the S form) obtained by the above mentioned procedures showed similar activity. In contrast, in the case of 3 other R-mutants the urea extracts were superior to the others in their ability to protect against following infection while in the case of another mutant the guanidine thiocyanate extract showed the highest protective capacity. In the case of the most active extracts the degree of protection was comparable to that obtained by immunizing with acetone-killed bacteria. The protective activity in the various extracts did not correlate with the content of lipopolysaccharide, phospholipid or nucleic acid. This poses the question of how far ubiquitous protein antigens play a role in immunity to gram-negative infections.