Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J‐5 lipopolysaccharide

Abstract

The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Escherichia coli J-5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial infections with gram-negative pathogens. The successive de-O-acylation and de-N-acylation of J-5 LPS yielded phosphorylated oligosaccharides which represent the complete carbohydrate backbone. Five compounds were separated by high-performance anion-exchange chromatography and analysed by one-dimensional and twodimensional homonuclear and heteronuclear 1 H-NMR, 13 C-NMR and 31 P-NMR spectroscopy. The main product was a nonasaccharide of the structurewherein all sugars are present as d-pyranoses. Hep and Kdo represent l-glycero-d-manno-heptose and 3-deoxy-d-manno-oct-2-ulosonic acid, respectively. In addition, two octasaccharides and two heptasaccharides were isolated that were partial structures of the nonasaccharide. In one octasaccharide the terminal a-d-GlcpN was missing and an additional phosphate group linked to O4 of the branched heptose was present, whereas in the other octasaccharide the sidechain Kdo was missing. In both heptasaccharides the side-chain a-d-GlcpN-(137)-l-a-d-Hepp-disaccharide was absent; they differed in their phosphate substitution. Whereas both heptasaccharides contained two phosphates in the lipid-A backbone (b-1,6-linked GlcpN-disaccharide at the reducing end) and one phosphate group at O4 of the first heptose, only one of them was additionally substituted with phosphate at O4 of the second heptose.Keywords: antibodies; biosynthesis; E. coli J-5; lipopolysaccharide; MALDI-TOF MS; phosphorylation; RcP + -chemotype.Lipopolysaccharide (LPS, endotoxin) is the major component of the outer membrane of gram-negative bacteria and is responsible for many of the pathophysiological effects observed during infections with gram-negative pathogens that may lead to septic shock and death [1,2]. Enterobacterial LPS consists of three domains, i.e. lipid A, core region and O-specific chain [1,3], of which lipid A is structurally the most conserved among different pathogenic bacteria [4] and represents the toxic principle of LPS [5]. Despite decades of intensive research and a great demand for alternatives, conventional antibiotic treatment still remains the main weapon of clinicians in the treatment of such infections. As the toxic effects exerted by LPS are independent of the viability of bacteria and considering the increasing resistance of pathogenic bacteria to antibiotics, the search for alternative strategies is of major importance. One of the most promising approaches for the immunotherapy of septic shock is passive immunization with antibodies that are directed against the conserved regions of LPS, i.e. lipid A and the core region. Such antibodies are expected to be cross-reactive with different gramnegative pathogens and might therefore be cross-protective. The induction of such antibodies could be achieved by immunization with lipid A presented in an appropriate form or mut...

Structural Analysis of Oligosaccharides from Lipopolysaccharide (LPS) of Escherichia coli K12 Strain W3100 Reveals a Link between Inner and Outer Core LPS Biosynthesis

“…9, and "Results." previously (9). Briefly, for semipreparative chromatography a gradient of 1-50% B in 60 min with eluents (A) H 2 O and (B) 1 M NaOAc was used.…”

“…Experimental evidence has been obtained recently showing that certain LPS structural motifs are recognized by outer membrane proteins (Omp) and that LPS plays a role for the correct folding and activity of proteins, such as PhoE (5), OmpT (6), and FhuA (7,8). The isolation and detailed structural characterization of all different glycoforms present in LPS from Escherichia coli J-5 aided the interpretation of results from in vitro folding experiments of outer membrane proteins (5,6) and furthermore gave an insight into the biosynthesis of LPS (9). Whereas the presence and distribution of negative charges is undoubtedly very important, the highly conserved nature of carbohydrates that build the inner core region implies that they are particularly suited to fulfill certain functions.…”

“…We have developed methods to purify the different glycoforms present in LPS (9) and for their conjugation to protein carrier molecules allowing their use in serological studies (25,26). The advantage of the developed procedures is the fact that they allow the determination of the relative distribution of the various glycoforms and allow the study of their immunoreactivities.…”

Identification of a Cross-reactive Epitope Widely Present in Lipopolysaccharide from Enterobacteria and Recognized by the Cross-protective Monoclonal Antibody WN1 222-5

“…Minnesota (Salmonella R1 core type) (17), and the E. coli strain J-5 (⌬galE mutant) (14) were cultivated, and LPS was extracted from each. Briefly, LPS was isolated by phenol/chloroform/petrolether extraction (18) and de-O-acylated by mild hydrazinolysis, followed by de-N-acylation under strong alkaline conditions (4 M KOH, 120°C, 16 h) (11).…”

“…1), were obtained by deacylation under strong alkaline conditions (14). The chemical structures of five different E. coli core types (R1 to R4 and K-12) and two chemically distinct core oligosaccharides of S. enterica (2,(15)(16)(17) are known, and all possess identical inner core structures (Fig.…”