Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats

Abstract

Haemophilus ducreyi, a gram-negative human mucosal pathogen, is one of the principal causes of genital ulcer disease. The lipooligosaccharides (LOS) of these bacteria are considered to be a major virulence factor and have been implicated in the adherence and invasion of H. ducreyi to several human cell types. An isogenic heptosyltransferase-III knockout strain (waaQ) was recently constructed from H. ducreyi 35000 wild-type strain and immunochemical and molecular weight data of the isolated LOS suggested the presence of poly-N-acetyllactosamine (LacNAc) (Filiatrault et al., Infect. Immun. 2000, 68, 3352-3361). In this present study, the structures of these novel LOS-glycoforms were characterized by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry in combination with exoglycosidase digestion. Detailed structural information was obtained for the oligosaccharide (OS) portions of these LOS showing between one to five linear LacNAc repeats on the non-reducing terminus of the main oligosaccharide branch. When grown on solid media, the organism produced LacNAc repeats that were further modified by the addition of sialic acid. Enzymatic digestion with ␤-galactosidase, ␤-N-acetylhexosaminidase, and neuraminidase type VI-A yielded truncated glycoforms consistent with a polyLacNAc structure capped at various end points with sialic acid. ESI-MS/MS mass spectrometry on a quadrupole time-offlight instrument was particularly effective in obtaining detailed structural information on the least abundant, high-mass glycoforms. Although LOS containing terminal di-LacNAc have been reported, this is the first time to our knowledge that a linear polyLacNAc structure has been characterized in bacteria. (J Am Soc Mass Spectrom 2002, 13, 724 -734)

Structural Characterization of an Oligosaccharide Made by Neisseria sicca

“…However, the number of disaccharide repeats was limited to two or three (10). Analogous results were generated when the infectivity of Haemophilus ducreyi, a causative agent of genital ulcers, was studied; infectious strains made LOS with a few lactosamine repeats (4,22).…”

“…Sandlin and Stein (21) identified a strain of Neisseria sicca that expressed an unusual glycolipid that appeared based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be analogous to LPS made by enteric bacteria. A poly-N-acetyllactosamine repeat was seen in some strains of the gonococcus (10), and we postulated that the repeating carbohydrate found in N. sicca could be a variant of this structure.A few studies have shown that both Neisseria and Haemophilus strains have the ability to extend their LOS by adding lactosamine repeats to form polylactosamine; these strains seem to possess increased virulence (4,10,22). When N. gonorrhoeae MS11mkC was used to inoculate healthy male volunteers, 100% of the volunteers developed urethritis, compared to an infectivity rate of 40% for strains expressing a truncated LOS (23).…”

“…A few studies have shown that both Neisseria and Haemophilus strains have the ability to extend their LOS by adding lactosamine repeats to form polylactosamine; these strains seem to possess increased virulence (4,10,22). When N. gonorrhoeae MS11mkC was used to inoculate healthy male volunteers, 100% of the volunteers developed urethritis, compared to an infectivity rate of 40% for strains expressing a truncated LOS (23).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 2001–2002

“…For example, Haemophilus ducreyi mutants lacking the enzymes heptosyltransferase III and b-(1 ! 4)-glucosyltransferase were shown to synthesize LOS with poly-Nacetyllactosamine repeats (Schilling et al, 2002a). The GlcNAc transferase from this species has been shown to be encoded by the lgtA gene (Sun et al, 2002).…”

“…These include membranes such as those having a low molecular weight cut-off that can be used for drop dialysis (Schilling et al, 2001(Schilling et al, , 2002aYlönen et al, 2001;Post et al, 2002); resins, such as SP20SS (Huang et al, 2002), OASIS cartridges from Waters (Charlwood et al, 2000(Charlwood et al, , 2001aCharlwood, Skehel, & Camilleri, 2001b) (for 3-acetamido-6-aminoacridine (AA-Ac, 2/5) derivatives), H þ Dowex AG 50W X8 (for acidic xylo-oligosaccharides) (Kabel, Schols, & Voragen, 2002b) or Amberlite IR-120 (H þ ) (Janusch et al, 2002), anion-exchange ZipTips (for O-deacylated lipooligosaccharides) (Schilling et al, 2002a), or C18 ZipTips (Charlwood et al, 2001a;Charlwood, Skehel, & Camilleri, 2001b) from Millipore for AA-Ac-derivatized N-linked glycans. Various investigators have desalted lipopolysaccharides using Dowex 50W (Schilling et al, 2001(Schilling et al, , 2002aJones et al, 2002). Graphatized carbon is also proving to be an effective clean-up medium (Mills et al, 2001a,c).…”