Lex2B, a Phase-Variable Glycosyltransferase, Adds either a Glucose or a Galactose to Haemophilus influenzae Lipopolysaccharide

Abstract

Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second ␤-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl-or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter-and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.Haemophilus influenzae is a gram-negative bacterium that is an obligate commensal in the human respiratory tract. This organism, however, has a propensity to spread contiguously, and the predominant unencapsulated (nontypeable H. influenzae [NTHi]) strains can cause diseases such as respiratory tract infections and otitis media (OM). Lipopolysaccharide (LPS), the major cell surface glycolipid, significantly influences hostmicrobe interactions and is a virulence determinant. From the well-conserved triheptose-containing inner core backbone, oligosaccharides (OS) comprising mainly hexose sugars and various nonsugar substituents extend, and these OS vary in composition between strains (31, 32, 36). The number of sugars and substituent groups comprising these OS can vary at a high frequency within individual strains, and the reversible loss and gain of epitopes is called phase variation (30). Phase variation of LPS in H. influenzae is mediated by switching in the expression of LPS-specific loci that contain tetranucleotide repeats (microsatellites) in the 5Ј end of the reading frame (17,19,21,38). These repeat tracts undergo mutation via DNA slippage during replication (25), leading to loss or gain of repeat units that place the reading frame in or out of frame with the translational initiation codon. Thus, depending on the number of tetranucleotide units in the tract, each phase-variable gene can be correctly translated ("on") or not correctly translated ("off"). For any H. influenzae strain having multiple phasevariable loci, a population of cells can simultaneously generate a range of LPS glycoforms, allowing the organism to adapt to different microenvironments of the host and helping it escape ...

Expression of a new disialyllacto structure in the lipopolysaccharide of non-typeable Haemophilus influenzae

“…The most variety of outer core extentions were observed from GlcI. Elongation at O-4 of GlcI is dependent on the phase variable lex2 locus as it was established in NTHi strain 1124 and 2019 ( Figure 5) [47,48]. It encodes either for a glucosyltransferase or a galactosyltransferase depending on the amino acid at position 157 [47].…”

Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20

“…The further addition of -Gal to -Gal is controlled by a phase variable gene lgtC (41). Notably, lex2 (55,56) is not only phase variable but also exists in two allelic variants. The presence of the phase-variable and host-mimicking digalactoside epitope on LPS increases resistance to naturally acquired bactericidal antibody in humans (57).…”

Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide

“…This result strongly suggests co-dependent transcription of the losA and losB genes and a polar effect of mutation of losA on losB expression. We predict that LosA would add a hexose as part of any further extension from HepIV, with LosA1 predicted to add a glucose and LosA2 a galactose based on sequence similarities to other glycosyltransferases (Deadman et al, 2006(Deadman et al, , 2009. We cannot confirm losA function in the relevant mutant strain as the HepIVcontaining acceptor glycoform is absent.…”