Identification of a lipopolysaccharide α‐2,3‐sialyltransferase from Haemophilus influenzae

Abstract

We have identified a gene for the addition of N‐acetylneuraminic acid (Neu5Ac) in an α‐2,3‐linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase‐variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N‐acetyl‐lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase‐variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non‐typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl‐α‐(2–3)‐lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an α‐2,3‐sialyltransferase activity, is the first reported phase‐variable sialyltransferase gene.

Sialic acid utilization by bacterial pathogens

“…The main sialyltransferase is Lic3A (Fig. 1), which adds a-2,3-Neu5Ac (Hood et al, 2001), and has been shown to be absolutely required for bacterial survival in the middle ear in an animal model of otitis media (Bouchet et al, 2003). Amongst the other sialyltransferases, the Lic3A-homologue Lic3B is a bifunctional enzyme that can add mono-or disialic acid to the LPS acceptor ( Fig.…”

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

“…The sialylation of LPS glycoforms by strain 486 depends only on the Lic3A sialyltransferase (7,17) and therefore inactivation of lic3A by insertion of a kanamycin resistance cassette (7) completely prevented expression of sialylated glycoforms. Isogenic sialic acid-deficient mutants of NTHi strains 162, 285, 375, 477, and 486 were constructed by mutating the gene encoding CMP-Neu5Ac synthetase (siaB) as described (6).…”

“…Isogenic sialic acid-deficient mutants of NTHi strains 162, 285, 375, 477, and 486 were constructed by mutating the gene encoding CMP-Neu5Ac synthetase (siaB) as described (6). We confirmed the genotype of all mutants by using PCR and Southern hybridization (6,7). The LPS phenotypes were confirmed as described (6).…”

“…We have reported that all NTHi OM isolates have the potential to incorporate sialic acid [N-acetylneuraminic acid (Neu5Ac)] into their lipopolysaccharide (LPS), and that strains expressing this sugar are more resistant to the bactericidal activity of normal human serum in vitro (6)(7)(8). Although sialylation of LPS has been implicated in bacterial virulence (9), the contribution of sialylated LPS glycoforms of Hi has not been investigated in vivo.…”

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

“…Many pathogenic bacteria, such as H. influenzae, N. gonorrhoeae , and N. meningitides , can decorate their LOS by sialylation, either by incorporating Neu5Ac into their LOS, cell membrane or capsule. Alternatively, some of the Escherichia coli strains and N. meningitidis produce capsular polysaccharides containing Neu5Ac [37]. The mechanism of LOS sialylation in these microbes differs.…”