Characterization of a trifunctional glucosyltransferase essential for Moraxella catarrhalis lipooligosaccharide assembly

Abstract

The human respiratory tract pathogen Moraxella catarrhalis expresses lipooligosaccharides (LOS), glycolipid surface moieties that are associated with enhanced colonization and virulence. Recent studies have delineated the major steps required for the biosynthesis and assembly of the M. catarrhalis LOS molecule. We previously demonstrated that the glucosyltransferase enzyme Lgt3 is responsible for the addition of at least one glucose (Glc) molecule, at the β-(1-4) position, to the inner core of the LOS molecule. Our data further suggested a potential multifunctional role for Lgt3 in LOS biosynthesis. The studies reported here demonstrate that the Lgt3 enzyme possesses two glycosyltransferase domains (A1 and A2) similar to that of other bifunctional glycosyltransferase enzymes involved in surface polysaccharide biosynthesis in Escherichia coli, Pasteurella multocida and Streptococcus pyogenes. Each Lgt3 domain contains a conserved DXD motif, shown to be involved in the catalytic activity of other glycosyltransferases. To determine the function of each domain, A1 (N-terminal), A2 (C-terminal) and double A1A2 site-directed DAD to AAA mutants were constructed and the resulting LOS phenotypes of these modified strains were analyzed. Our studies indicate that the Lgt3 N-terminal A1 catalytic domain is responsible for the addition of the first β-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the β-(1-4) Glc and the β-(1-6) Glc, confirming the bifunctional nature of this domain. The results from these experiments demonstrate that Lgt3 is a novel, multifunctional transferase responsible for the addition of three Glcs with differing linkages onto the inner core of M. catarrhalis LOS.

Keywords: Moraxella catarrhalis; glucosyltransferase; lipooligosaccharide; trifunctional.

Fig. 1.

(A) The relative positions of the two putative catalytic DAD motifs in the Lgt3 polypeptide and the two GT-2 domains as predicted by sequence homology to known β-glycosyltransferases and bifunctional glycosyltransferase enzymes. (B) Alignment of the amino acid sequences of the two domains; the aspartic acid residues of the DAD motifs mutated to alanines are marked with asterisks. Shading denotes identical (dark) and similar (light) amino acid residues.

Fig. 2.

A silver-stained SDS–PAGE gel depicting the LOS profiles of wild-type 7169 (lane 1), 7169::lgt3KH (lane 2) and the site-directed mutant constructs Lgt3A1 (lane 3), Lgt3A2 (lane 4) and Lgt3A1A2 (lane 5).

Fig. 3.

Composite of a silver-stained SDS–PAGE gel (A) and the corresponding western blot probed with MAb 3F7 (B) evaluating LOS profiles of wild-type strain 7169 (lane 1), Lgt3A1 (lane 2), Lgt3A1 + plgt3A2 (lane 3), Lgt3A2 (lane 4), Lgt3A2 + plgt3A1 (lane 5), Lgt3A1A2 (lane 6), Lgt3A1A2 + plgt3 (lane 7) demonstrating restoration of wild-type phenotype following in trans complementation of the site-directed mutants with the corresponding functional catalytic domain(s).

Fig. 4.

Schematic depicting the biochemical structure of the wild-type M. catarrhalis serotype B LOS molecule. All the three LOS serotypes contain the common core OS indicated in gray. The functions of the glycosyltransferases involved in LOS biosynthesis are indicated. The trifunctional catalysis mediated by Lgt3 was determined in this study.