A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019

Abstract

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri- l- glycero- d- manno-heptosyl inner-core moiety ( l-α- d-Hep p-(1→2)-[ PEtn→6]- l-α- d-Hep p-(1→3)-[β- d-GlcI p-(1→4)]- l-α- d-Hep p-(1→5)-α-Kdo p) to which addition of β- d-Glc p to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a β- d-Gal p is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of β- d-Gal p-(1→4-β- d-Glc p-(1→ from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMS n on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β- d-Glc p extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β- d-Gal p to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.