The Caenorhabditis elegansbus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance

Abstract

Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegans bus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 ␤1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydraterich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species.

Glycosylation Genes Expressed in Seam Cells Determine Complex Surface Properties and Bacterial Adhesion to the Cuticle of Caenorhabditis elegans

“…Examination of these animals confirmed the seam-cell expression and revealed significant expression in the posterior intestine, as well as in head muscles (Figure 2,D and E). The intestinal expression is consistent with detectable alterations in intestinal lectin staining, which are described elsewhere (Palaima et al 2010).…”

“…Scrutiny of the possible coding sequence for bus-2/ K08D12.5 revealed an anomaly, as noted elsewhere (Palaima et al 2010): the protein sequence based on the gene model in WormBase (referential data freeze WS200) can be improved in terms of alignment with other glycosyltransferases, including the Caenorhabditis briggsae ortholog of bus-2, by replacing the 19-aa WormBase exon 3 with a different 43-aa exon 3. The region concerned (exon 2 to exon 5) was amplified from a C. elegans cDNA preparation, and sequencing demonstrated the presence of the 43-aa exon 3 and its predicted junction with exon 4.…”

“…Probable biochemical properties of BUS-2 are reported at greater length elsewhere (Palaima et al 2010). It is predicted to be a galactosyltransferase, and plausible orthologs can be found in the genomes of all Caenorhabditis species so far sequenced (see WormBase).…”

“…but tests on existing bus-2 mutants revealed that they had a Bah phenotype as well as a Bus phenotype (Darby et al 2007). Genetic mapping together with analysis of transposon-induced mutations of bus-2 established that bus-2 corresponds to K08D12.5, encoding a predicted galactosyltransferase enzyme (Palaima et al 2010). Sequencing of EMS-induced alleles revealed missense changes in all cases (Figure 1).…”

“…The second of these, e2776, has therefore been used in an extensive characterization of altered glycosylation patterns in bus-2 worms (Palaima et al 2010). The EMS alleles are all missense changes in conserved regions of the predicted protein; the existing bus-2 reference allele, e2687, causes a Cys-to-Tyr change in one of these conserved regions and has been used for most of this work as a presumed null or severe hypomorph.…”