Identification and characterization of the N-acetylglucosamine glycosyltransferase gene of Haemophilus ducreyi

Abstract

Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted ulcerative disease. In the present study, the Neisseria gonorrhoeae lgtA lipooligosaccharide glycosyltransferase gene was used to identify a homologue in the genome of H. ducreyi. The putative H. ducreyi glycosyltransferase gene (designated lgtA) was cloned and insertionally inactivated, and an isogenic mutant was constructed. Structural studies demonstrated that the lipooligosaccharide isolated from the mutant strain lacked N-acetylglucosamine and distal sugars found in the lipooligosaccharide produced by the parental strain. The isogenic mutant was transformed with a recombinant plasmid containing the putative glycosyltransferase gene. This strain produced the lipooligosaccharide glycoforms produced by the parental strain, confirming that the lgtA gene encodes the N-acetylglucosamine glycosyltransferase.

FIG. 1.

Structure of the LOS of H. ducreyi 35000HP. The nomenclature for the A, a, and b branches, as well as each glycoform has been previously reported (4).

FIG. 2.

Structure of the lgtA region of the strain 35000HP genome and the cloned amplicon containing the lgtA gene (pRSM2378). A DNA fragment containing the lgtA gene was amplified by PCR and cloned into the PCRBlunt II-TOPO vector. The unique BamHI site was used for insertional inactivation of the lgtA gene.

FIG. 3.

SDS-PAGE and Western blot analysis of LOS preparations. (A) LOS preparations from 35000HP (lane 1), the lgtB mutant (lane 2), the lgtA mutant (lane 3), and the lbgA mutant (lane 4) were prepared by the microphenol technique, separated by SDS-PAGE, and visualized by silver staining. The corresponding structure for each glycoform is shown in Fig. 1. (B) Silver-stained SDS-polyacrylamide gel and the corresponding Western blot. The gel contains LOS preparations from H. ducreyi 35000HP (lane 1), 35000HP-RSM212, the lgtA mutant (lane 2), and the complemented mutant, 35000HP-RSM212/pRSM2391 (lane 3). The blot, containing the same preparations, was probed with MAb 3F11. Glycoform A5 and glycoform A5b2 react with MAb 3F11. The A5b2 glycoform in the LOS preparation from strain 35000HP was too low in concentration to be observed in this blot.

FIG. 4.

Linear negative-ion MALDI-TOF spectra of the O-deacylated LOS preparations from strains 35000HP (a) and 35000HP-RSM212 (b). The observed molecular ions for the individual LOS species appear as unresolved clusters whose centroids correspond to the average mass. The peaks at m/z 1,532.2 that are labeled with an asterisk were found to be artifacts. All Kdo residues were replaced with phosphorylphosphoethanolamine.