Biosynthesis of Sialylated Lipooligosaccharides in Haemophilus ducreyi Is Dependent on Exogenous Sialic Acid and Not Mannosamine. Incorporation Studies Using N-Acylmannosamine Analogues, N-Glycolylneuraminic Acid, and ¹³C-Labeled N-Acetylneuraminic Acid

Abstract

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.

Haemophilus parasuis α-2,3-sialyltransferase-mediated lipooligosaccharide sialylation contributes to bacterial pathogenicity

“…In H. parasuis , the lsgB gene product, α-2,3-sialyltransferase, sialylates the terminal galactose of LOS by using 5ʹ-cytidinemonophospho-N-acetylneuraminic acid or other donors [20]. Moreover, H. influenzae sialylates its LOS by a precursor scavenging mechanism [40], and an analogous strategy has also been reported in H. ducreyi [41] and H. somnus [42]. These observations indicate that LOS sialylation is an important modification in members of the Haemophilus genus.…”

Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides

“…Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS was performed on a Voyager DE mass spectrometer as described (18). The samples were further analyzed by electrospray ionization MS (ESI-MS) on a quadrupole orthogonal time-of-flight mass spectrometer (QSTAR, MDS Sciex) equipped with a Protana nanospray ion source (18).…”

“…The samples were further analyzed by electrospray ionization MS (ESI-MS) on a quadrupole orthogonal time-of-flight mass spectrometer (QSTAR, MDS Sciex) equipped with a Protana nanospray ion source (18). Mass spectra (ESI-MS) and tandem mass spectra (ESI-MS͞MS) of O-deacylated LOS were recorded in positive ion mode, and calibrated as described previously (18).…”

“…The samples were further analyzed by electrospray ionization MS (ESI-MS) on a quadrupole orthogonal time-of-flight mass spectrometer (QSTAR, MDS Sciex) equipped with a Protana nanospray ion source (18). Mass spectra (ESI-MS) and tandem mass spectra (ESI-MS͞MS) of O-deacylated LOS were recorded in positive ion mode, and calibrated as described previously (18). Additional internal calibration by using well characterized O-LOS glycoforms with exact molecular masses M ϭ 2956.0004 ([M ϩ 3H] 3ϩ at m͞z 986.3413) and M ϭ 2793.9476 ([M ϩ 3H] 3ϩ at m͞z 932.3237) yielded mass accuracies of Ϯ10 ppm.…”

“…We have previously investigated the biosynthetic origin of sialyl-LOS in H. ducreyi (18). Unlike mammalian cells, which biosynthesize the majority of their sialic acid from the committed precursor N-acetylmannosamine (ManNAc), H. ducreyi must import sialic acid from the extracellular environment; sialyl-LOS are generated exclusively from exogenous sialic acid that has been scavenged, converted to CMP-NeuAc, and transferred onto LOS by a sialyltransferase (Fig.…”