D-090. Identification and Characterization of a Glycosyltransferase Gene Essential for the Synthesis of Moraxella catarrhalis Lipooligosaccharides

J. M. Schwingel1, F. St. Michael2, A. D. Cox2, H. Masoud2, J. C. Richards2, A. A. Campagnari1;
1Univ. at Buffalo, Buffalo, NY, 2Natl. Res. Council of Canada, Ottawa, ON, CANADA.

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory infections in adults suffering from chronic obstructive pulmonary disease (COPD). The M. catarrhalis lipooligosaccharide (LOS) molecule is classified into three serotypes related to its structure. Previous studies have shown that strains expressing either a serotype A LOS (60%) or a serotype B LOS (30-35%) account for the majority of clinical isolates. M. catarrhalis LOS is unique among most organisms in that the initial carbohydrate residue attached to the lipid A-KDO2 molecule is a glucose, whereas normally enteric bacteria have a heptose residue in this position. The function of LOS glycosyltransferase genes (lgt1, lgt2, lgt3, lgt4, and lgt5) involved in the assembly of the LOS molecule for all M. catarrhalis LOS serotypes have been previously identified by our laboratory and others. Despite these studies the enzyme responsible for the initial glucose addition to the Lipid A-KDO2 core was not identified. In this study, the gene encoding the enzyme required for that initial glucose addition for LOS assembly has been identified by nucleotide and amino acid similarities to putative glycosyltransferases in other bacteria in all three M. catarrhalis LOS serotypes. This gene, lgt6, is located in a region of the chromosome distinct from the other previously characterized lgts. The Lgt6 glycosyltransferase is unique among described glycosyltransferases in its KDO-glucose linkage. Deletion mutants of lgt6 were constructed in all three LOS serotypes and their LOS structures were analyzed by SDS-PAGE and electrospray mass spectrometry to assign function. Mass spectrometry results indicated a lipid A-KDO2 LOS structure, suggesting that lgt6 functions as the initial lgt adding an α(1-5)-linked glucose to the KDO. Reversion of these mutants with a wild-type copy of lgt6 restored full-length LOS. Further biologic assays show the importance of a full-length LOS molecule to M. catarrhalis pathogenesis.

166/D. Immune Responses to Pathogenic Bacteria

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