Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats.
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 beta-galactosidase, beta-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-of-flight 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.
Schilling
Birgit
B
Buck Institute for Age Research, Novato, California 94945, USA.
Gibson
Bradford W
BW
Filiatrault
Melanie
M
Campagnari
Anthony A
AA
D000606:Amino Sugars / Q000737:chemistry*; D015153:Blotting, Western; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry; D006191:Haemophilus ducreyi / Q000737:chemistry*; D006868:Hydrolysis; D008070:Lipopolysaccharides / Q000737:chemistry*; D008970:Molecular Weight; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000606:Amino Sugars; D003470:Culture Media; D008070:Lipopolysaccharides; C032233:lactosamine; D006026:Glycoside Hydrolases