Cross-Reactions Between E. coli 0 Antigens and Antigens Outside the Enterobacteriaceae Group .669

Das Makromolekulare Kolloquium fand vom 11. bis 13. Marz 1965 in Freiburg/Brsg. statt und wurde vom Institut fur Makromolekulare Chemie der Universitat Freiburg/Brsg. veranstaltet.

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure 32-␣-Man- (132) (18,55), and the O3-and O5-specific polysaccharides of Klebsiella strains are identical to the O8 and O9 polysaccharides of E. coli (8,18,29). Mannans of algal origin were found to exert antitumor activity (37). Such an activity could later be attributed also to the mannan-containing LPS of E. coli and Klebsiella strains (13, 37).The genetics of LPS biosynthesis in enteric bacteria is well documented in recent reviews (33,43,48,55). Two mechanisms, block and monomeric, have been described for O-polysaccharide synthesis (49). In the block mechanism, observed for Salmonella typhimurium and related Salmonella serotypes, the oligosaccharide repeating units are assembled on undecaprenol phosphate (antigen carrier lipid [ACL]) under the direction of rfb genes. The first sugar transferred was found to be galactose-1-phosphate, and the corresponding transferase gene was termed rfbP (48). The repeating units are polymerized under the direction of the rfc gene, which may be located outside of or within the rfb gene cluster (41). The chain length is controlled by the rol gene, located between gnd and his (3, 4). The monomeric mechanism, experimentally proven only for E. coli O8 and O9 (18, 55), consists of the direct and sequential transfer of the monosaccharide residues from their nucleotideactivated precursors to the nonreducing end of the growing polysaccharide chain.The synthesis of some O polysaccharides requires the rfe gene. According to this requirement, LPS biosynthesis can also be divided into rfe-dependent and rfe-independent pathways. The rfe gene, first described by Mäkelä et al. (31), was found to be essential for the synthesis of the O polysaccharide in Salmonella strains of O groups C1 and L, and E. coli O8 and O9 (18, 33) and more recently in E. coli O4, O7, O18, O75, and O111 (1, 23a). It was reported to determine the tunicamycinsensitive transfer of N-acetylglucosamine (GlcNAc)-1-phosphate from UDP-GlcNAc to undecaprenol monophosphate

The capsular polysaccharide was isolated from Escherichia coli 010 : K5 : H4; it could not be obtained from a uncapsulated ( K Y ) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1 : 1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-fi-glucuronyl-I ,4-a-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin.It has been shown previously that pathogenicity of Escherichia coli bacteria correlated with the chemical and physical properties of their surface polysaccharide antigens [I, 21. E. coli strains with certain capsular polysaccharide antigens (K antigens) are frequently associated with urinary tract infections, especially in children [2 -41. The K antigens which are most prominent in these extraintestinal infections are KI, K2, K3, K5, K12 and K13. Encapsulated bacteria give rise to antibodies against K antigens in man and experimental animals. However, antibodies against the K5 polysaccharide antigen are only very rarely found after immunization with E. coli exhibiting the K5 antigen. It was therefore desirable to elucidate the structure of the K5 antigen in the hope that structural considerations may help to explain the poor immunogenicity of this important bacterial surface antigen.The following structural information of the above-mentioned K antigenic capsular polysaccharides of E. coli is available : the K1 antigen is a homopolysaccharide consisting of cc-2,S-linked neuraminic acid [5], the K2 antigen is a teichoicacid-like polymer consisting of galactopyranosyl-glycerol phosphate and galactofuranosyl-glycerol phosphate units in a molar ratio of 2: 1 [6] and the K13 antigen is a heteropolysaccharide with a repeating sequence of 3-linked ribose and 7-linked 2-keto-3-deoxy-manno-octulosonic acid (dOclA) [7]. In preliminary studies we have found that the K12 antigen consists of L-rhamnose and dOclA in a molar ratio of 2: 1 (unpublished results). In this publication we report the structure of the KS antigen from E. coli 010 : K5 : H4, compare it with similar bacterial polysaccharides and discuss the low immunogenicity of this capsular polysaccharide. MATERIALS AND METHODS Bacteria and CultivationEscherichia coli strain Bi 8337-41 (010 : K5 : H4) was used. It was obtained by Drs I. and F. Qrskov of the International Abbreviation. dOclA, 2-kelo-3-deoxy-~-manno-octulosonic acid Enzyme. b-Glucuronidase (EC 3.2.1.31).Escherichia Centre, Copenhagen. The growth conditions for the isolation of the capsular polysaccharide have been described previously [6,7]. Isolation and Purijication qf the Capsular PolysaccharideThe acidic capsular polysaccharide and the bacterial cells were precipitated from the liquid cultures by the additi...

Lipopolysaccharide preparations from R (rough) Escherichia coli 0 8 -, SR (semirough) Salmonella typhimurium and S (smooth) strains E. coli 0 8 and Citrobacter 396 were disintegrated with sodium dodecylsulfate and subjected to polyacrylamide gel electrophoresis in the presence of 1 % sodium dodecylsulfate. The results obtained were compared with those obtained from the same lipopolysaccharide preparations by degradation analysis. In dodecylsulfate gel electrophoresis the lipopolysaccharide preparation from the E. coli R mutant and the S. typhimurium SR mutant showed one band each (R-and SR-band, respectively) with different electrophoretic mobilities. The lipopolysaccharide preparation from the E. coli 0s-strain exhibited two bands, one of which had the same electrophoretic mobility as the R-band and the other was identified as S-band. The lipopolysaccharide preparation from the Citrobacter 396 S-strain exhibited four bands: one R-band, one SR-band and two S-bands. The results showed that wild-type S strains contain more than one type of lipopolysaccharide. They differ in the length of their 0-specific polysaccharide chains. The lipid A content of the different lipopolysaccharide was expressed in their electrophoretic mobilities.For the characterization of enterobacterial lipopolysaccharides we have been using mild acid hydrolysis (removal of lipid A) and fractionation of the polysaccharide moiety by gel permeation chromatography [l -31. The carbohydrate moiety of the lipopolysaccharides from most smooth (S) bacteria can be separated on Sephadex into two main fractions, one representing the core oligosaccharide substituted with the 0-specific polysaccharide and the other representing the unsubstituted core oligosaccharide (for definitions see . More recently, we found that in some cases the fractionation pattern was more complex. Besides unsubstituted core oligosaccharide (R) and core oligosaccharide substituted with the O-specific polysaccharide (S) we have also found core oligosaccharide substituted with only one 0-specific repeating unit. Other separations revealed the presence of core oligosaccharides substituted with 0-specific polysaccharides of different chain lengths. Thus, bacteria may exhibit on their surface several types of lipopolysaccharides. The heterogeneity was only observed after degradation of the lipopolysaccharide preparations and fractionation of the degraded polysaccharides. Because these procedures are time consuming, we have sought for a simpler method to analyze the molecular state (S-, SR-, R-forms) of lipopolysaccharides in bacterial wild types and mutants. We have found that electrophoresis of lipopolysaccharides in polyacrylamide gels in the presence of the dissociating surfactant sodium dodecylsulfate is a simple and useful tool for the molecular characterization of enterobacterial lipopolysaccharides. MATERIALS AND METHODS Bacteria and CultivationEscherichia coli 0 8 K27-: H-(F 492) and the R mutant derived from it (F 470), a Salmonella typhimurium SR strain (F 971) and Citrobacte...