lgtC Expression Modulates Resistance to C4b Deposition on an Invasive Nontypeable Haemophilus influenzae

Abstract

We have previously shown that C3 binding to serum-resistant nontypeable Haemophilus influenzae (NTHi) strain R2866 is slower than C3 binding to a serum-sensitive strain. Ab-dependent classical pathway activation is required for complement-dependent killing of NTHi. To further characterize the mechanism(s) of serum resistance of R2866, we compared binding of complement component C4b to R2866 with a serum-sensitive variant, R3392. We show that C4b binding to R2866 relative to R3392 was delayed, suggesting regulation of the classical pathway of complement. Increased C4b deposition on R3392 was independent of the amount and subclass of Ab binding, suggesting that an impediment to C4b binding existed on R2866. Immunoblotting and mass spectrometry indicated that lipooligosaccharide and outer membrane proteins P2 and P5 were targets for C4b. P2 and P5 sequences and expression levels were similar in both strains. Insertional inactivation of the phase-variable lipooligosaccharide biosynthesis gene lgtC in R2866 augmented C4b deposition to levels seen with R3392 and rendered the bacteria sensitive to serum and whole blood. These results suggest a direct role of lgtC expression in the inhibition of C4b deposition and consequent serum resistance of R2866. Alteration of surface glycans of NTHi may be a critical event in determining the ability of a strain to evade host defenses and cause disseminated infection.

Interaction with C4b-Binding Protein Contributes to Nontypeable Haemophilus influenzae Serum Resistance

“…By using two independent methods, we showed that most of the NTHi strains bound C4BP, whereas the majority of the typeable strains did not. This finding is in contrast to a recent study, in which no binding of H. influenzae to C4BP was found (46). However, this study focused on one particular strain of NTHi: R3392.…”

Binding of Complement Regulators to Invasive Nontypeable Haemophilus influenzae Isolates Is Not Increased Compared to Nasopharyngeal Isolates, but Serum Resistance Is Linked to Disease Severity

“…Phase variation of outer membrane proteins and lipooligosaccharides also contributes to virulence of H. influenzae and is involved in evasion of the immune system (39). Intriguingly, phase variation can affect binding of regulatory proteins and has been shown to affect serum resistance (19). Since phase variation is a common phenomenon and depends on the current bacterial environment, it cannot be excluded that phase variation is a factor affecting the in vitro results of our study.…”

“…Importantly, no significant difference in survival between the various clinical strains from blood samples and those from nasopharynxes was found when serum resistance was determined, suggesting the need for NTHi, irrespective of isolation site, for resistance of bactericidal activity of human serum. Previous studies showed that the NTHi strain R2866, which was isolated from a child with meningitis, had a high degree of serum resistance depending on the expression of the lipooligosaccharide biosynthesis gene lgtC (9,19,40). The phase-variable lgtC expression was demonstrated to inhibit C4b deposition and render the bacteria more resistant to human serum (19).…”

“…Previous studies showed that the NTHi strain R2866, which was isolated from a child with meningitis, had a high degree of serum resistance depending on the expression of the lipooligosaccharide biosynthesis gene lgtC (9,19,40). The phase-variable lgtC expression was demonstrated to inhibit C4b deposition and render the bacteria more resistant to human serum (19). Phase variation of outer membrane proteins and lipooligosaccharides also contributes to virulence of H. influenzae and is involved in evasion of the immune system (39).…”

Relationships of Nontypeable Haemophilus influenzae Strains to Hemolytic and Nonhemolytic Haemophilus haemolyticus Strains

“…The lic2A and lgtC genes were nearly ubiquitous in H. influenzae (Ն90%) but were only 16 and 2% prevalent, respectively, in H. haemolyticus. Interestingly, the combined expression of these genes in H. influenzae is necessary to form a LOS digalactoside structure (34,35) that antigenically mimics the pK blood group of humans and increases the serum resistance of the organism by inhibiting the deposition of the C4b released from the classical complement pathway (21,61). Of the 17 H. haemolyticus strains in this study that possessed a lic2A gene, none possessed the lgtC gene (unpublished data), suggesting that the digalactoside structure is not made in H. haemolyticus.…”