No abstract

The chromosomal genes rfaC and rfaF of Escherichia coli W3110 were inactivated by allelic-replacement mutagenesis to generate a defined strain lacking both heptosyltransferases which catalyze in lipopolysaccharide (LPS) biosynthesis the transfer of the first two L-glycero-D-manno-heptose (Hep) residues to 3-deoxy-~-mannm-~-octu~oson~c acid (Kdo). The LPS of the mutant was isolated and its chemical structure was investigated by compositional analysis and nuclear magnetic resonance spectroscopy of isolated, deacylated oligosaccharide phosphates. The basic structure was a tetrasaccharide n-Kdo-(2+4)-a-Kdo-(2+6)-P-~-GlcN4P-( 1+6)-a-~-GlcNl P which in LPS was substituted at position 0 7 of Kdo I1 by 2-aminoethanol phosphate in non-stoichiometric amounts. 2-Aminoethanol was cleaved during deacylation of the LPS by successive hydrazinolysis and KOH treatment and, in addition, phosphate migration from 0 7 to 0 8 of Kdo I1 occurred. Thus, the oligosaccharides u-Kdo7P -(2-4)-a-Kdo-(2-6)-P-~- GlcN4P-( 1+6)-a-~-GlcNlP and ~-K~O~P-(~+~)-~-K~O-(~+~)-P-D-GICN~P-( 1+6)-a-~-GlcN1P could be isolated. KOH treatment of the two trisphosphates and authentic methyl 3-deoxy-D-manno-octulopyranoside 7-(2-acetamidoethyl phosphate) proved that phosphate migration only took place when the phosphate group was substituted with 2-aminoethanol. Complementation studies with plasmid-encoded rjaC and rfaF genes revealed that the mutant strain can be used in combination with LPS-specific antibodies for the cloning and characterization of heptosytransferases which glycosylate Kdo residues of the inner core region of LPS.

The lipopolysaccharide (LPS) of Chlamydia trachomatis serotype E was isolated from tissue culture-grown elementary bodies and analyzed structurally by mass spectrometry and 1 H, 13 C and 31 P nuclear magnetic resonance. The LPS is composed of the same pentasaccharide bisphosphate aKdo-(2-8)-aKdo-(2-4)-aKdo-(2-6)-bGlcN-4P-(1-6)-aGlcN-1P (Kdo is 3-deoxy-a-D-manno-oct-2-ulosonic acid) as reported for C. trachomatis serotype L 2 [Rund, S., Lindner, B., Brade, H. and Holst, O. (1999) J. Biol. Chem. 274, 16819-16824]. The glucosamine disaccharide backbone is substituted with a complex mixture of fatty acids with ester or amide linkage whereby no ester-linked hydroxy fatty acids were found. The LPS was purified carefully (with contaminations by protein or nucleic acids below 0.3%) and tested for its ability to induce proinflammatory cytokines in several readout systems in comparison to LPS from C. trachomatis serotype L 2 and Chlamydophila psittaci strain 6BC as well as enterobacterial smooth and rough LPS and synthetic hexaacyl lipid A. The chlamydial LPS were at least 10 times less active than typical endotoxins; specificity of the activities was confirmed by inhibition with the LPS antagonist, B1233, or with monoclonal antibodies against chlamydial LPS. Like other LPS, the chlamydial LPS used toll-like receptor TLR4 for signalling, but unlike other LPS activation was strictly CD14-dependent.

Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-α-d-manno-heptose (l,d-heptose), but not by 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic α1→3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-α-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.