The role of a repetitive DNA motif (5′‐CAAT‐3′) in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope αGal(1–4)βGal

Abstract

Haemophilus influenzae lipopolysaccharide (LPS) contains structures, defined by monoclonal antibodies, which undergo phase variation. This investigation reports the nucleotide sequence of lic2A, which is required for the expression of at least three phase-variable LPS epitopes, one of which has the structure alpha Gal(1-4)beta Gal. lic2A contains multiple tandem repeats of the tetramer 5'-CAAT-3'. Previous studies have correlated changes in the number of 5'-CAAT-3' repeats with the phase-variable expression of the alpha Gal(1-4)beta Gal epitope. To obtain direct evidence for this, the 5'-CAAT-3' repeat region from lic2A was amplified directly from immunostained colonies and sequenced. This demonstrated that the variable expression of LPS epitopes, including alpha Gal(1-4)beta Gal, is in part directly dependent upon the number of copies of 5'-CAAT-3' within lic2A.

Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyl transferases.

“…The laboratories of Moxon and Hansen (28)(29)(30) have shown that this also occurs in another mucosal pathogen, Haemophilus influenzae, where at least four genes are subject to phase variation. In this organism, the mechanism is also by slipped-strand mispairing due to repeated tetrameric sequences, which can be either CAAT or GCAA.…”

The Edwardsiella ictaluri O polysaccharide biosynthesis gene cluster and the role of O polysaccharide in resistance to normal catfish serum and catfish neutrophils

“…The motifs, FXFFD located 30-40 residues from the N terminus and EDD located 90 residues from the N terminus, were identified in WbiB. WbiB is also similar to Lic2A, which is required for production of the phase-variable aGal-1,4-bGal epitope in Haemophilus influenzae type B lipooligosaccharide (High et al, 1993), and to Haemophilus ducreyi lipooligosaccharide biosynthesis enzyme LbgA, which is a b-1,4-galactosyltransferase (Tullius et al, 2002).…”

The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

“…For direct sequencing of 59-GCAA repeat tracts in lex2A, DNA fragments generated by PCR amplification with a biotinylated form of primer lex2for and primer lex39 (Table 2) were denatured in 150 mM NaOH and single-stranded DNA was purified by the use of streptavidin-coated Dynabeads (Dynal) as described by High et al (1993). The DNA obtained was sequenced by the dideoxy chain termination method using a modified T7 polymerase (Sequenase II kit: United States Biochemical Corp.) and primer lex2seq (Table 2).…”

“…Tetranucleotide-repeat-containing loci in H. influenzae are involved in the phase-variable expression of a digalactoside (lic2A and lgtC) (High et al, 1993;Hood et al, 1996), of phosphorylcholine (ChoP) (lic1) (Weiser et al, 1997) and of sialic acid (lic3A) (Hood et al, 2001) in the LPS. A fifth locus, lex2, comprising two reading frames lex2A and lex2B, which is absent from the published Rd genome sequence (Fleischmann et al, 1995), was identified in a serotype b strain, DL42 (Jarosik & Hansen, 1994).…”

“…This reversible loss and gain of epitopes is known as phase variation. Phase variation of LPS in H. influenzae is mediated by 'on-off ' switching of the translation of LPS-specific loci through tetranucleotide repeat tracts within the 59 end of the reading frame (Weiser et al, 1989a, b;High et al, 1993;Hood et al, 1996;Jarosik & Hansen, 1994). The number of repeat units within a tract can change due to mis-pairing of complementary strands at the repeat region during DNA replication, resulting in loss or gain of one or more repeat units and a switch in the reading frame to be translated (Levinson & Gutman, 1987).…”