Structural studies of lipooligosaccharides from Haemophilus ducreyi ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence for intrastrain heterogeneity with the production of mutually exclusive sialylated or elongated glycoforms

Schweda, Elke K. H.; Jonasson, Jon; Jansson, Per‐Erik

doi:10.1128/jb.177.18.5316-5321.1995

Cited by 37 publications

(40 citation statements)

References 24 publications

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“…In addition, Melaugh et al [10] reported that a di-LacNAc glycoform was also present at very low abundances in strain 35000. A similar observation was reported by Schweda et al [14] for several clinical isolates. The di-LacNAc pathway appears to compete with a more dominant biosynthetic pathway that adds sialic acid, effectively capping the extension of the oligosaccharide branch [10].…”

supporting

confidence: 76%

“…To obtain LOS-glycoforms that are more suitable to ESI-MS/MS under positive-ion conditions, the O-deacylated LOS glycoforms were treated with aqueous HF (4°C, 12 h) 2), which gave structural proof that a terminal sialic acid residue was added to the terminal LacNAc moiety. The presence of the abundant sialylated di-LacNAc species (A' 5 b 2 a 1 ) was of considerable interest, as the di-LacNAc species has been reported to be present in wild type strains but not the sialylated counterpart [14]. To obtain unambiguous data for this sialylated diLacNAc glycoform, an ESI-MS/MS spectrum was re- …”

Section: Resultsmentioning

confidence: 99%

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Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats

Schilling

Gibson

Filiatrault

et al. 2002

J. Am. Soc. Mass Spectrom.

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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)

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supporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats

Schilling

Gibson

Filiatrault

et al. 2002

J. Am. Soc. Mass Spectrom.

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“…Several studies have demonstrated that H. ducreyi LOS cause ulcers in rabbits and mice (9,21,55). Recent structural studies have begun to define the LOS glycoforms expressed by several different H. ducreyi strains, including strains 35000, ITM5535, ITM3147, and ACY1 (27,29,45). In addition, recent data have demonstrated that the principal LOS glycoform expressed by most H. ducreyi strains is highly sialylated on the terminal galactose residue of N-acetyllactosamine in a manner similar to that previously reported for Neisseria gonorrhoeae (27; reviewed in reference 39).…”

mentioning

confidence: 79%

“…GC-MS of the partially permethylated alditol acetates established the presence of four sugars: a terminal glucose and terminal heptose, a 2-linked heptose, and a 3,4-linked heptose. The absence of 2-keto-3-deoxyoctulosonic acid (Kdo) was expected due to the conversion of the presumed 4-phospho-Kdo sugar to the anhydro-Kdo products during the mild-acid step (6) as reported previously for several Haemophilus species (34,35,45,46), including the LOS from the parental H. ducreyi 35000 (28,29). These reducing terminal anhydro-Kdo diastereomers are presumably destroyed during the acid hydrolysis conditions used in the monosaccharide analysis.…”

Section: Identification Of Tn916 Mutants Deficient In Los Biosynthesismentioning

confidence: 99%

Characterization of a transposon Tn916-generated mutant of Haemophilus ducreyi 35000 defective in lipooligosaccharide biosynthesis

et al. 1997

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“…In order to increase our understanding of the role of LOS in the pathogenesis of chancroid, the structures of the major glycoforms of the LOS from H. ducreyi strains have been determined (8,28,29,41,42). The major glycoform from strain 35000HP has the following structure: Gal␤134GlcNAc␤13 3Gal␤134Hep␣136Glc␤13(Hep␣132Hep␣13)3,4Hep␣13 5Kdo.…”

mentioning

confidence: 99%

Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of Haemophilus ducreyi

et al. 2000

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Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.Chancroid is a genital ulcerative disease caused by Haemophilus ducreyi which is prevalent in several developing countries (30, 46). Although chancroid has been characterized histologically (16, 17), little is known about the molecular basis for ulcer formation. A number of putative virulence determinants of H. ducreyi have been described, including a hemolytic cytotoxin (3,35,36,45), cytolethal distending toxin (11), pili (9), a hemoglobin binding protein (15,44), and lipooligosaccharide (LOS) (10,18,25,32). The structure of the oligosaccharide portion of the LOS resembles the structure of human paraglobosides. Thus, it has been proposed that the LOS may help the organism evade the human immune response by molecular mimicry (26, 27). Direct injection of H. ducreyi LOS causes ulcers in rabbits and mice (10, 25, 47). Injection of H. ducreyi LOS causes a stronger inflammatory response in rabbits than injection of rough lipopolysaccharide from Escherichia coli (10). Other studies have implicated LOS in adherence to fibroblasts...

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Structural studies of lipooligosaccharides from Haemophilus ducreyi ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence for intrastrain heterogeneity with the production of mutually exclusive sialylated or elongated glycoforms

Cited by 37 publications

References 24 publications

Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats

Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats

Characterization of a transposon Tn916-generated mutant of Haemophilus ducreyi 35000 defective in lipooligosaccharide biosynthesis

Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of Haemophilus ducreyi

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