Modified lipooligosaccharide structure protects nontypeable Haemophilus influenzae from IgM-mediated complement killing in experimental otitis media

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM.

Importance: Nontypeable Haemophilus influenzae (NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such as Neisseria meningitidis, Moraxella catarrhalis, and Bordetella pertussis.

FIG 1

Determination of the complement resistance and IgM binding of clinical NTHi isolates. (A) Survival of MEF (n = 22) and NPS (n = 24) isolates was determined in 5% normal human serum and expressed as percent survival compared to that in 5% heat-inactivated human serum for 60 min. (B) Serum IgG and IgM binding on 25 serum-sensitive and 21 complement-resistant NTHi isolates was determined by flow cytometry. Statistical significance was determined with an unpaired t test with Welch’s correction. *, P < 0.05; **, P < 0.01. (C) Correlation between complement resistance and IgM binding.

FIG 2

The R2866_0112 gene deletion mutant exhibits decreased complement resistance. (A) Relative expression of R2866_112 mRNA was analyzed by qRT-PCR in 25 serum-sensitive and 21 complement-resistant clinical isolates Statistical significance was determined with an unpaired t test with Welch’s correction. (B) Complement resistance of R2866, Δ0112 mutant, and ΔlgtC mutant (n = 4) was determined with 40% NHS or 40% heat-inactivated NHS. Statistical significance was determined with a two-way analysis of variance and the Bonferroni post hoc test. (C) Complement resistance of R2866, Δ0112 mutant, Δ1813 mutant, Δ1095-1101 mutant, Δ0112/Δ1813 mutant, and Δ0112/Δ1095-1101 mutant was determined in 40% NHS (n = 4). Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test. (D to G) Complement resistance of Rd (HI0461) (D), 86-028NP (NTHI0592) (E), 3655 (CGSHi3655_02894) (F), and 1521062 (G) was determined with 10% (Rd), 20% (3655), or 40% (86-028NP and 1521062) serum, respectively (n = 3). Statistical significance was determined on log₁₀-transformed data with a one-way analysis of variance and the Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

FIG 3

The R2866_0112 gene deletion mutant expresses an altered LOS structure. (A) LOS analysis of R2866, Δ0112 mutant, and ΔlgtC mutant strains by Tris-Tricine SDS-PAGE and silver staining. (B and C) Outer membrane stability of R2866 wild type and mutant as determined by sensitivity to EDTA (n = 5) (B) or polymyxin B (n = 8) (C). Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test or a with a two-way analysis of variance and the Bonferroni post hoc test, respectively. OD₆₂₀, optical density at 620 nm; NS, not significant.

FIG 4

IgM binds R2866Δ0112 mutant LOS directly. (A) Serum IgG and IgM binding on R2866 and Δ0112 mutant as determined by flow cytometry (n = 9). Statistical significance was determined with an unpaired t test with Welch’s correction. (B) Complement resistance of R2866 and Δ0112 mutant strains was determined in heat-inactivated NHS (HI-NHS), NHS, or IgM-depleted serum (n = 4). Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test. (C) Correlation between Δ0112 mutant serum survival and IgM binding. (D and F) Direct binding of IgM to LOS was analyzed by silver staining (loading control) and Western blotting. (E) Relative IgM binding to LOS of R2866, Δ0112, and ΔlgtC mutant was calculated (n = 3). (G) Signal intensities in arbitrary units (AU) of IgM binding to LOS of clinical isolates were calculated (n = 4). Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test or with an unpaired t test. *, P < 0.05; **, P < 0.01; NS, not significant.

FIG 5

The R2866_0112 gene mutant shows decreased virulence in a murine coinfection otitis media model. Mice were inoculated with 10^4.5 PFU of influenza A virus (IAV) or mock treated 3 days before intranasal infection with 5 × 10⁷ CFU of NTHi. (A and B) CFU counts in the nose (A) or the middle ears (B) were determined 48 and 96 h postinfection (n = 10). (C, D, F, and G) Mice were infected with 10^4.5 PFU of IAV 3 days before intranasal infection with a 1:1 ratio of NTHi 1521062 wild type (WT) and the R2866_0112 mutant (5 × 10⁷ CFU total). CFU counts in the nose (C and D) or the middle ears (F and G) were determined 48 and 96 h postinfection (n = 10). Statistical significance was determined with a Mann-Whitney test. (E and H) CI scores were calculated. Statistical significance was determined with a one-way analysis of variance and the Tukey post hoc test. **, P < 0.01; ***, P < 0.001.