Mass Spectral Characterization of Lipooligosaccharides from<i>Haemophilus influenzae</i>2019

Gaucher, Sara P.; Cancilla, Mark T.; Phillips, Nancy J.; Gibson, and Bradford W.; Leary, Julie A.

doi:10.1021/bi001181k

Cited by 35 publications

(26 citation statements)

References 44 publications

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“…The isolated LOS was O deacylated by treatment with anhydrous hydrazine and analyzed by MALDI-TOF mass spectrometry. Previous data showed that NTHI strain 2019 produces a complex mixture of LOS glycoforms when it is grown in culture medium (12,40 Table 2), which is characteristic of H. influenzae LOS (28,40). In the present study, when strain 2019 was grown on solid medium supplemented with Neu5Ac, its LOS repertoire expanded to include new sialylated, disialylated, and polysialylated species (Fig.…”

Section: Nthi Biofilm Formationsupporting

confidence: 55%

Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N -Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

et al. 2004

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Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate ␣-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with ␣2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were twoto fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing ␣2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

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Section: Nthi Biofilm Formationsupporting

confidence: 55%

Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N -Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

et al. 2004

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“…Previous studies have shown that NTHi 2019 produces a complex mixture of LOS glycoforms (7,9,20). The major component of this mixture has been extensively studied and is known to consist of a lactose moiety (Gal␤1,4-Glc␤1-) linked in a ␤1,4 linkage to Table 2) gave a similar repertoire of glycoforms as that seen previously by MALDI-TOF-MS (9), with the major glycoform being the lactose-containing glycoform modified with 2 or 3 phosphoethanolamine (PEA) moieties (B 2 and B 3 , respectively).…”

Section: Resultsmentioning

confidence: 99%

Novel Sialic Acid Transporter of Haemophilus influenzae

et al. 2005

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Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.

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“…Dissociation techniques applied to FT-ICR MS analysis of biomolecules include sustained off-resonance collisionally-activated dissociation [20 -22] (SORI-CAD), infrared multiphoton dissociation [23,24] (IRMPD), electron capture dissociation [25,26] (ECD), and electron detachment dissociation [27] (EDD). SORI-CAD has been employed to characterize lipooligosaccharides from the pathogen Haemophilus influenzae [28] and galactosylceramide from pig intestine [29]. ECD has been used to localize sites of O-glycosylation [30] and fatty acid posttranslational modifications in peptides [31].…”

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confidence: 99%

Structural characterization of the GM1 ganglioside by infrared multiphoton dissociation, electron capture dissociation, and electron detachment dissociation electrospray ionization FT-ICR MS/MS

McFarland¹,

Marshall²,

Hendrickson³

et al. 2005

J. Am. Soc. Mass Spectrom.

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Gangliosides play important biological roles and structural characterization of both the carbohydrate and the lipid moieties is important. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), electron detachment dissociation (EDD), and infrared multiphoton dissociation (IRMPD) provide extensive fragmentation of the protonated and deprotonated GM1 ganglioside. ECD provides extensive structural information, including identification of both halves of the ceramide and cleavage of the acetyl moiety of the N-acetylated sugars. IRMPD provides similar glycan fragmentation but no cleavage of the acetyl moiety. Cleavage between the fatty acid and the long-chain base of the ceramide moiety is seen in negative-ion IRMPD but not in positive-ion IRMPD of GM1. Furthermore, this extent of fragmentation requires a range of laser powers, whereas all information is available from a single ECD experiment. However, stepwise fragmentation by IRMPD may be used to map the relative labilities for a series of cleavages. EDD provides the alternative of electron-induced fragmentation for negative ions with extensive fragmentation, but suffers from low efficiency as well as complication of data analysis by frequent loss of hydrogen atoms. We also show that analysis of MS/MS data for glycolipids is greatly simplified by classification of product ion masses to specific regions of the ganglioside based solely on mass defect graphical analysis. (J Am Soc Mass Spectrom 2005, 16, 752-762)

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Mass Spectral Characterization of Lipooligosaccharides fromHaemophilus influenzae2019

Cited by 35 publications

References 44 publications

Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N -Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N -Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

Novel Sialic Acid Transporter of Haemophilus influenzae

Structural characterization of the GM1 ganglioside by infrared multiphoton dissociation, electron capture dissociation, and electron detachment dissociation electrospray ionization FT-ICR MS/MS

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