Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

Abstract

Moraxella catarrhalis isolates express lipooligosaccharide (LOS) molecules on their surface, which share epitopes similar to that of the Neisseria and Haemophilus species. These common LOS epitopes have been implicated in various steps of pathogenesis for the different organisms. In this study, a cluster of three LOS glycosyltransferase genes (lgt) were identified in M. catarrhalis 7169, a strain that produces a serotype B LOS. Mutants in these glycosyltransferase genes were constructed, and the resulting LOS phenotypes were consistent with varying degrees of truncation compared to wild-type LOS. The LOS structures of each lgt mutant were no longer detected by a monoclonal antibody (MAb 4G5) specific to a highly conserved terminal epitope nor by a monoclonal antibody (MAb 3F7) specific to the serotype B LOS side chain. Mass spectrometry of the LOS glycoforms assembled by two of these lgt mutants indicated that lgt1 encodes an ␣(1-2) glucosyltransferase and the lgt2 encodes a ␤(1-4) galactosyltransferase. However, these structural studies could not delineate the function for lgt3. Therefore, M. catarrhalis lgt3 was introduced into a defined ␤(1-4) glucosyltransferase Haemophilus ducreyi 35000glu؊ mutant in trans, and monoclonal antibody analysis confirmed that Lgt3 complemented the LOS defect. These data suggest that lgt3 encodes a glucosyltransferase involved in the addition of a ␤(1-4)-linked glucose to the inner core. Furthermore, we conclude that this enzymatic step is essential for the assembly of the complete LOS glycoform expressed by M. catarrhalis 7169.Moraxella catarrhalis is a gram-negative human respiratory pathogen that causes 15 to 20% of acute otitis media in children (56). In addition, this bacterium is responsible for 10 to 35% of lower respiratory infections in adults with chronic obstructive pulmonary disease, the fourth leading cause of death in the United States (20). The emergence of M. catarrhalis as an important human pathogen has occurred in the past 15 years due to the increasing prevalence of ␤-lactamase-positive strains, the high incidence of recurrent infections despite successful antibiotic treatment, and the lack of an effective vaccine (10,39,50,56). Another factor that likely contributes to the persistence of M. catarrhalis disease is the lack of understanding of the basic bacterial factors and mechanisms that promote colonization and survival in the host.Although there have been a number of putative virulence factors described for M. catarrhalis, the actual role of these components in pathogenesis remains largely undefined (13,19,20,31,53). One of the most prominent surface components expressed in the outer membrane of this bacterium is the lipooligosaccharide (LOS) (12). The LOS of M. catarrhalis is similar to the lipopolysaccharide (LPS) of other gram-negative organisms, but it lacks a repeating O antigen (21). Previous studies have suggested that LOS is important for the pathogenesis of other respiratory pathogens, such as Neisseria meningitidis and Haemophilus influenzae, aid...

Multiplex PCR Assay That Identifies the Major Lipooligosaccharide Serotype Expressed by Moraxella catarrhalis Clinical Isolates

“…1A and C. A ClustalW alignment with the translated sequence of this open reading frame (Lgt4) in both strains revealed 46% identity and 60% similarity to Lgt1, an ␣(1-2) glucosyltransferase in M. catarrhalis 7169 (6). The 5Ј end of this cluster in serotype A and C strains contained DNA sequence that is homologous to the 5Ј region of the lgt cluster in 7169, as depicted in all three clusters in Fig.…”

“…A number of putative virulence factors have been described for M. catarrhalis (8,11,12,17,20), including a surface-exposed lipooligosaccharide (LOS) (6,18,23,32). Structural and serological studies with M. catarrhalis have described only three different LOS serotypes (termed A, B, and C), which vary in length and content of the oligosaccharide branches (3-5, 13, 15, 28).…”

“…Recently, a cluster of three glycosyltransferase (lgt) genes were identified and characterized in a strain of M. catarrhalis 7169 expressing serotype B LOS (6). Primers 406 and 408 (Table 1) designed to this region were subsequently used in PCRs with chromosomal DNA from M. catarrhalis 25238, the previously defined LOS serotype A strain, and M. catarrhalis RS10, the previously defined LOS serotype C strain (Table 2) (3,4).…”

Characterization of the Six Glycosyltransferases Involved in the Biosynthesis of Yersinia enterocolitica Serotype O:3 Lipopolysaccharide Outer Core

“…Furthermore, according to these results, the Glcp(I) residue has to be added by WbcL before WbcK is able to add the Glcp(II) residue. Similar strict order of GTase functions has been shown for the growing lipooligosaccharide molecule of Moraxella catarrhalis; addition of specific sugar residues is critical to the enzymes that act further downstream in the biosynthesis of the full-length lipooligosaccharide molecule (38).…”

An Unusual Carbohydrate Conformation is Evident in Moraxella catarrhalis Oligosaccharides

“…Depending on the serotype, up to an additional 2-3 hexose residues may be present on the 1-4 and 1-6 linked chains ( Figure 1). Recently, we [14,15] and others [16][17][18][19] have begun to unravel the roles of the various glycosyltransferases that direct the serotype specific biosynthesis of the LOS in M. catarrhalis.…”