Sialic Acid Protects Nontypeable Haemophilus influenzae from Natural IgM and Promotes Survival in Murine Respiratory Tract

Abstract

Nontypeable Haemophilus influenzae (NTHi), a common inhabitant of the human nasopharynx and upper airways, causes opportunistic respiratory tract infections that are frequently recurring and chronic. NTHi utilizes sialic acid from the host to evade antibacterial defenses and persist in mucosal tissues; however, the role of sialic acid scavenged by NTHi during infection is not fully understood. We previously showed that sialylation protects specific epitopes on NTHi lipooligosaccharide (LOS) targeted by bactericidal IgM in normal human serum. Here, we evaluated the importance of immune evasion mediated by LOS sialylation in the mouse respiratory tract using wild-type H. influenzae and an isogenic siaB mutant incapable of sialylating the LOS. Sialylation protected common NTHi glycan structures recognized by human and murine IgM and protected NTHi from complement-mediated killing directed by IgM against these structures. Protection from IgM binding by sialylated LOS correlated with decreased survival of the siaB mutant versus the wild type in the murine lung. Complement depletion with cobra venom factor increased survival of the siaB mutant in the nasopharynx but not in the lungs, suggesting differing roles of sialylation at these sites. Prior infection increased IgM against H. influenzae but not against sialic acid-protected epitopes, consistent with sialic acid-mediated immune evasion during infection. These results provide mechanistic insight into an NTHi evasive strategy against an immune defense conserved across host species, highlighting the potential of the mouse model for development of anti-infective strategies targeting LOS antigens of NTHi.

Keywords: Haemophilus influenzae; IgM; NTHi; complement resistance; immune evasion; lipooligosaccharide; lung infection; mouse model; natural antibody; sialic acid.

FIG 1

LOS diagram of NTHi 375 and serum IgM targets. The diagram is based on structural information described elsewhere (5) and previously determined targets of IgM in human serum (7). Glc, glucose; Gal, galactose; Neu5Ac, N-acetylneuraminic acid; GalNAc, N-acetylgalactosamine; PC, phosphorylcholine; Hep, heptose; Kdo, 2-keto-3-deoxyoctulosonic acid. Competition between Lic3A and LgtC is indicated by dashed line. Lic3B is a bifunctional α-2,3- and α-2,8-sialyltransferase present in some NTHi strains (indicated by dashed brackets), including 375.

FIG 2

Sialylation protects NTHi LOS epitopes from bactericidal IgM in mouse serum. Heat-inactivated C57BL/6 mouse serum was preabsorbed with NTHi 375 wild type (WT) and an isogenic Δlic2A mutant grown on MIc^SA−. (A) Diagram depicting the differences in LOS structures on HepIII between these two strains and the IgM antibodies expected to remain in preabsorbed sera. The indicated galactose residues (βGal and αGal) are within the terminal βGal and αGal structures (t-βGal and t-αGal) which contain IgM epitopes. Also shown are corresponding antibodies to both epitopes predicted to remain in Δlic2A^SA− preabsorbed serum. Neither of these IgM species is predicted to remain in WT^SA− preabsorbed serum. (B) 375 WT grown on MIc^SA± was incubated with each preabsorbed serum (serum^abs) and IgM binding was measured by flow cytometry. Binding is depicted as a percentage of the maximum, calculated by normalizing the median fluorescence intensity (MFI) values of triplicate samples to the highest MFI. Statistical significance was evaluated by two-way ANOVA with Bonferroni’s multiple-comparison test. (C) 375 WT and the lgtC′ mutant were grown on MIc^SA± and incubated with each preabsorbed serum (Serumabs). Percent survival is the ratio of the number of CFU recovered from serum^abs-treated samples at 30 min to the number of input CFU at time zero. Means for triplicate samples are shown. The dashed line indicates the lower limit of detection at approximately 0.07%. Statistical significance of log-transformed survival ratios was evaluated by one-way ANOVA with Bonferroni’s multiple-comparison test (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not statistically significant).

FIG 3

Human and mouse serum share similar antibody specificities for LOS epitopes on NTHi 375. IgM binding to 375 wild type (WT) versus lgtC′ (A and B), and WT versus lgtC′ lic2A^OFF, a lic2A phase-off variant in the lgtC′ mutant background (C and D), all grown on MIc^SA±, were analyzed by ELISA (absorbance read at 620 nm) after incubation with human (A and C) and mouse (B and D) serum preabsorbed with 375 WT and the Δlic2A mutant grown on MIc^SA− (serum^abs for all at a final dilution of 1:25). lic2A is phase-on for the WT and lgtC′ strains and phase-off in the lgtC′ lic2A^OFF strain. IgM binding is shown as a percentage of the maximum normalized absorbance values, with the means and standard errors of the means (SEM) of triplicate samples graphed. Statistical significance was evaluated by one-way ANOVA with Bonferroni’s multiple-comparison test (****, P < 0.0001; **, P < 0.01; *, P < 0.05; ns, not statistically significant).

FIG 4

Immunization induces cross-reactive IgM to H. influenzae. Blood was collected from naive (n = 3) and immune (n = 3) mice on day 11 after immunization. Serum was obtained and heat inactivated before incubating with wild-type (WT) H. influenzae Rd and the siaB′ mutant grown on sBHI to measure IgM binding by flow cytometry. MFI values were log transformed [Y = log(y)] and normalized to get the percentage of the maximum, and means and SEM are shown. Statistical significance was evaluated by one-way ANOVA with Bonferroni’s multiple-comparison test (****, P < 0.0001; ***, P < 0.001; **, P < 0.01).

FIG 5

Sialic acid enhances in vivo survival of H. influenzae. (A) Mice received intranasal inoculations of Rd wild type (WT) or siaB′ mutants, each mixed with equal numbers of CFU of a SiaB⁺ competitor strain, RdLacZ (LacZ⁺), on day 14 postimmunization. Lung CFU were enumerated 20 h postinfection, and the ratio of the indicated experimental (LacZ⁻) strains to the reference strain (LacZ⁺) was calculated. The ratios, or competitive indices, are shown. Lines indicate geometric means. Statistical significance was evaluated by two-way ANOVA with Bonferroni’s multiple-comparison test (****, P < 0.0001). (B) Mice were treated with cobra venom factor (CVF) or PBS and inoculated intranasally with an equal mixture of RdsiaB′ and RdLacZ. Lungs and nasal septa were collected 22 h postinfection and plated for CFU determination. Shown are competitive index ratios at each site, calculated as for panel A. Lines indicate geometric means. Statistical significance was evaluated via unpaired, one-tailed t test (*, P < 0.05; ns, not statistically significant).

FIG 6

Deletion of siaB increases sensitivity of H. influenzae to mouse serum antibody-directed killing by complement. Survival of Rd wild type (WT) and a siaB mutant grown on MIc^SA following incubation with or without 10% heat-inactivated mouse serum (MS^Δi) as a source of antibody and 3% IgG/IgM antibody-depleted human complement active serum (HC) as a source of complement at 37°C for 30 min. Heat-inactivated HC (HC^Δi) was used as a control for complement-mediated killing. Percent survival is the ratio of the number of CFU recovered at 30 min relative to the input CFU at 0 min of incubation. Limit of detection was 0.2%. Log-transformed survival ratios were evaluated by one-way ANOVA with Bonferroni’s multiple-comparison test (***, P < 0.001; **, P < 0.01; ns, not statistically significant). The means for duplicate samples are shown.