Digalactoside Expression in the Lipopolysaccharide of Haemophilus influenzae and Its Role in Intravascular Survival

Abstract

Digalactoside (gal␣-1-4 gal␤) structures of the lipopolysaccharide (LPS) of Haemophilus influenzae are implicated in virulence. A confounding factor is that tetranucleotide repeats within the lic2A, lgtC, and lex2 genes mediate phase-variable expression of the digalactosides. By deleting these repeats, we constructed recombinant strains of RM153 constitutively expressing either one or two LPS digalactosides. Expression of two digalactosides, rather than one, was associated with increased virulence of H. influenzae in vivo.Lipopolysaccharide (LPS) is one of the major virulence determinants of the human pathogen Haemophilus influenzae. The number of hexoses and phosphate groups replacing the triheptose (HepI to HepIII) backbone ( Fig. 1) is variable within any strain owing to high-frequency translational switching (phase variation) of LPS genes containing repeat tracts. Phase-variable LPS genes include lic2A and lgtC, which are involved in the assembly of gal␣-1-4 gal␤ into the oligosaccharide extensions from the conserved triheptose backbone (7, 10), and lex2, which is involved in completion of the HepIattached diglucoside acceptor for this digalactoside ( Fig. 1) (4). The investigation of the association between digalactoside expression on H. influenzae LPS and virulence has relied on the monoclonal antibody (MAb) 4C4 for detecting the expression of the digalactoside (1, 2, 12, 13). The findings from these studies have been difficult to interpret because of the confounding factor of phase variation and the different numbers and locations of the digalactoside on oligosaccharide extensions in different strains (7,17,18).Recently, it has been shown that MAb 4C4 binds digalactosides that are part of the extensions from both HepI and HepII of the triheptose LPS backbone (Fig. 1) (4, 5). A type b clinical strain, RM153, used in previous virulence studies (9, 16), typically generates LPS molecules containing only four hexose sugars (17), depending on whether lic2A, lgtC, or lex2 is out of frame (4,7,10). This is in contrast to the related strain RM7004, which has up to nine hexose sugars in its LPS due to these three loci being predominantly in frame (Fig. 1). Knowing the LPS structure, the genes required for digalactoside assembly, and the fact that tetranucleotide repeats mediate phase variation, we constructed recombinant strains of RM153 in which variable expression of digalactosides was eliminated by removing the repeat tracts located within each of these genes. Briefly, two isogenic strains were constructed by transformation (6) using appropriate chromosomal DNA or plasmid constructs in which the repeats had been deleted for lic2A (8), lex2 (5), or lgtC. All three genes were constitutively expressed in the first strain, RM153lic2AϩtlgtCϩlex2ϩk, to facilitate expression of two digalactosides, while in the second strain, RM153lic2AϩtlgtCϩlex2Ϫk, lex2 was mutated such that only a single digalactoside in the extension from HepII was expressed (Fig.

Outer Membrane Protein P5 Is Required for Resistance of Nontypeable Haemophilus influenzae to Both the Classical and Alternative Complement Pathways

“…The outer surface LOS structures are critical mediators of serum resistance of H. influenzae that could potentially be altered by P5 (27,28,57,58). By silver staining of SDS-PAGE gels, the LOS bands were found to have similar mobility between Rd, RP5G, and RP5X or between NTV and NTP5V (see Fig.…”

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

“…Adding the second glucose in the extension from HepI of the LPS inner core backbone, as seen for Lex2B in serotype b strains in previous studies (14,21) and NTHi strain 1008 in our study, facilitates further oligosaccharide extension that includes digalactoside-containing moieties (Gal-␣-1 to 4-Gal-␤-1 to 4-Glc-␤-1 to 4-Glc-␣) that bind MAbs 4C4 and 5G8 (13,14,22). The presence of these digalactoside moieties can have biological consequences, and several studies have shown that the digalactoside on H. influenzae LPS can enhance the ability of the organism to escape the bactericidal activity of serum in in vitro assays and facilitates intravascular survival in the infant rat model of H. influenzae infection (4,10,12,19,22,23,39). The importance to the bacterium of varying the expression of the Gal-Gal-Glc moiety added to GlcI can be inferred from the fact that addition of each of these three consecutive sugars is dependent upon the action of an independent phase variably expressed glycosyltransferase (12).…”

“…The lex2 locus permits further OS extensions that include a digalactoside that binds MAb 5G8 (13) (Fig. 1) and that play a role in resistance of the bacteria to the killing effect of serum (12).…”

Identification of the Functional Initiation Codons of a Phase-Variable Gene of Haemophilus influenzae , lic2A , with the Potential for Differential Expression

“…Expression of this epitope, therefore, was examined using monoclonal antibodies specific for this epitope and both wild-type and mutant strains. PV of lgtC and lex2 was controlled for in our experiments by examining strains lacking repeats (11) or by checking that repeat numbers of these genes had not altered (data not shown). Analysis in RM7004 demonstrated that strong expression of the digalactoside epitope was associated with only the x/y frame of lic2A (Fig.…”

“…Expression of the digalactoside epitope is, in addition to lic2A, also dependent on the phase-variable genes lgtC and, to a lesser extent, lex2 (12, 16, 18). Thus, in addition to RM7004, the plasmid constructs were also transformed into an isogenic mutant (RM7004lgtC⌬5ЈGACA) in which expression of the lgtC gene is constitutive due to the absence of the tetranucleotide repeat tract (11).…”