Duplicate Copies oflic1Direct the Addition of Multiple Phosphocholine Residues in the Lipopolysaccharide ofHaemophilus influenzae

Abstract

The genes of the lic1 operon (lic1A to lic1D) are responsible for incorporation of phosphocholine (PCho) into the lipopolysaccharide (LPS) of Haemophilus influenzae. PCho plays a multifaceted role in the commensal and pathogenic lifestyles of a range of mucosal pathogens, including H. influenzae. Structural studies of the LPS of nontypeable H. influenzae (NTHI) have revealed that PCho can be linked to a hexose on any one of the oligosaccharide chain extensions from the conserved inner core triheptosyl backbone. In a collection of NTHI strains we found several strains in which there were two distinct but variant lic1D DNA sequences, genes predicted to encode the transferase responsible for directing the addition of PCho to LPS. The same isolates were also found to express concomitantly two PCho residues at distinct positions in their LPS. In one such NTHI isolate, isolate 1158, structural analysis of LPS from lic1 mutants confirmed that each of the two copies of lic1D directs the addition of PCho to a distinct location on the LPS. One position for PCho addition is a novel heptose, which is part of the oligosaccharide extension from the proximal heptose of the LPS inner core. Modification of the LPS by addition of two PCho residues resulted in increased binding of C-reactive protein and had consequential effects on the resistance of the organism to the killing effects of normal human serum compared to the effects of glycoforms containing one or no PCho. When bound, C-reactive protein leads to complement-mediated killing, indicating the potential biological significance of multiple PCho residues.Phosphocholine (PCho) has been detected on the cell surface of a range of bacteria, predominantly bacteria residing in the human respiratory tract, such as Haemophilus influenzae (5,25,39). In H. influenzae, PCho is a substituent of the lipopolysaccharide (LPS) molecule (24,25,39), the main glycolipid on the bacterial cell surface that is a target for host immune responses and influences both the commensal and pathogenic behavior of the organism (38). H. influenzae LPS comprises a membrane-anchoring lipid A (endotoxin) linked to oligosaccharide chains that extend from the bacterial cell surface. The LPS of a number of different strains have been analyzed and have been shown to be composed of a common L-glycero-Dmanno-heptose-containing inner-core trisaccharide unit attached to the lipid A via a phosphorylated 2-keto-3-deoxyoctulosonic acid (Kdo) residue (20, 23). Each of the heptose (Hep) residues can provide a point for addition of a hexose (Hex) residue, which in turn can lead to oligosaccharide chain extension (for a review, see reference 28). The nature of the oligosaccharide chains and the degree to which they contain noncarbohydrate substituents, such as PCho, show intra-and interstrain variation that can affect the virulence of the organism. PCho in H. influenzae LPS plays a role in persistence of the bacterium on the mucosal surface of the nasopharynx, at least in part by mediating bacterial adherence to, and invasi...

Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications

“…The addition of ChoP has been previously observed in a number of NTHi strains and can occur at any of the three core heptose (Hep) units (Fig. 4) (41,(44)(45)(46)(47)(48). These findings are in contrast to the most prevalent glycoforms expressed by NTHi strain 2019 (see Fig.…”

“…In NTHi, all three core heptoses can be further substituted with oligosaccharide chains, free phosphate (P), phosphoethanolamine (PEA), ChoP, acetate groups, and glycine (13,(41)(42)(43)(44)(45)(46)(47)(48). The addition of these various moieties can create highly heterogeneous LOS structures both between strains and within a strain.…”

Haemophilus influenzae and the complement system

“…CRP is an acute phase reactant that activates the classical pathway by binding C1q [14]. The level of ChoP expressed on the surface of NTHi correlates with the amount of CRP bound to NTHi, and CRP facilitates complement-mediated killing of the pathogen [50]. Furthermore, ChoP expression is associated with more efficient colonization of the nasopharynx in an experimental infant rat model [49].…”

Microbial Modulation of Host Immunity with the Small Molecule Phosphorylcholine

“…The licD allele carried by a given strain determines the location of attachment to the LPS (38). Finally, some strains of H. influenzae contain a duplication of the lic locus, resulting in the attachment of two ChoPs per LPS molecule (39).…”