Identification of a Bifunctional Lipopolysaccharide Sialyltransferase in Haemophilus influenzae

Abstract

The lipopolysaccharide (LPS) of non-typeableHere we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both ␣-2,3-and ␣-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance.

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

“…C. diphtheriae, L. monocytogenes, M. gallisepticum, S. pyogenes, S. agalactiae, and S. mutans all produce sialidases to remove sialic acid from host glycoproteins in order to uncover host adhesion receptors (108). The enzymatically released sialic acids are then used either as an energy source or as building blocks for incorporation into the bacterial cell envelope (108)(109)(110)(111)(112)(113), where they contribute to serum resistance (114). For N. meningitidis, H. influenzae, and C. jejuni, the sialylation of the cell envelope was found to be an important virulence factor that strongly contributed to the ability of these pathogens to adhere to, invade, and translocate across epithelial cells and to evade host immune responses (115)(116)(117)(118)(119).…”

Identification and Characterization of a Lipopolysaccharide ,2,3-Sialyltransferase from the Human Pathogen Helicobacter bizzozeronii

“…1). Clade A includes monofunctional ␣,2,3-STs and bifunctional ␣,2,3/ ␣,2,8-STs from Campylobacter jejuni (CstI [11], CstII [12], and CstIII [16]) and Haemophilus influenzae (Lic3A [23] and Lic3B [13]) as well as the ␣,2,3-STs from Pasteurella multocida (PM1174 [49]) and Helicobacter mustelae (HMU06130 [41] To detect amino acid positions potentially involved in the functional change, conserved sites of GT-42 STs were evaluated on the basis of structural data available for C. jejuni CstII (12) and CstI (11) and compared to the phylogenetic data. All the sequences belonging to clade A, both catalytic sites (Arg129 and His188 [CstII enumeration]), as well as the CMP-binding sites (Asn31, Ser132, Tyr156, and Tyr162) are conserved.…”

Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

“…Finally, lic2A encodes a glycosyltransferase that adds a Gal as part of a digalactose epitope (11). The LOS of Haemophilus can also contain sialic acid (5-acetylneuraminic acid [Neu5Ac]) as a terminal sugar residue; siaB encodes a CMP-Neu5Ac synthetase which catalyzes the formation of CMP-Neu5Ac, a nucleotide sugar donor used by the sialyltransferases encoded by lic3A, lic3B, siaA, and lsgB (12)(13)(14)(15).…”