Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

Abstract

Otitis media, a common and often recurrent bacterial infection of childhood, is a major reason for physician visits and the prescription of antimicrobials. Haemophilus influenzae is the cause of approximately 20% of episodes of bacterial otitis media, but most strains lack the capsule, a factor known to play a critical role in the virulence of strains causing invasive H. influenzae disease. Here we show that in capsule-deficient (nontypeable) strains, sialic acid, a terminal residue of the core sugars of H. influenzae lipopolysaccharide (LPS), is a critical virulence factor in the pathogenesis of experimental otitis media in chinchillas. We used five epidemiologically distinct H. influenzae isolates, representative of the genetic diversity of strains causing otitis media, to inoculate the middle ear of chinchillas. All animals developed acute bacterial otitis media that persisted for up to 3 wk, whereas isogenic sialic acid-deficient mutants (disrupted sialyltransferase or CMP-acetylneuraminic acid synthetase genes) were profoundly attenuated. MS analysis indicated that WT bacteria used to inoculate animals lacked any sialylated LPS glycoforms. In contrast, LPS of ex vivo organisms recovered from chinchilla middle ear exudates was sialylated. We conclude that sialylated LPS glycoforms play a key role in pathogenicity of nontypeable H. influenzae and depend on scavenging the essential precursors from the host during the infection.

Fig. 1.

Phylogenic tree based on ribotyping of 542 Hi strains. Five isolates (162, 285, 375, 477, and 486) were selected to infect the chinchillas. Independent confirmation of this dendrogram was obtained based on capsular operon gene polymorphism analysis (15) and congruence of a ribotype-based phylogenetic tree of a subset of the type a–f isolates with results from multilocus enzyme electrophoresis analysis (16). Further validation was obtained by comparative gene sequencing of recA and 16S ribosomal RNA genes among a representative subset of 50 of the isolates and multilocus sequence typing analysis (12) of a representative set involving 51 of the NT isolates.

Fig. 2.

Summary of viable counts (log₁₀) of Hi, y axis, from cultures of fluid obtained over a period of 26 days (x axis) after inoculation of 50–100 Hi at day 0. Open circles represent quantitative cultures from individual samples of middle-ear fluid after inoculation with one of five representative Hi strains (see Fig. 1): 486, 375, 477, 285, or 162. Filled circles indicate the results of chinchillas inoculated with the isogenic siaB mutants. The lowest detectable number of organisms is 100 cfu/ml (logarithm 100 = 2). Thus, for all samples obtained from chinchillas where organisms were not detected (zero cfu), a value of log₁₀₀ was assigned, and these data points are plotted within the area labeled “Less than 100 cfu/ml.”

Fig. 3.

Hi LPS comprises a heterogeneous mixture of glycoforms consisting of an oligosaccharide moiety attached to a membrane anchoring lipid A component. (A) Structural model of the conserved inner core region of the oligosaccharide portion of the molecule. (B) Space-filling molecular model of the minimum energy conformer of the sialyllactose containing LPS glycoform of NTHi strain 375 calculated by a Monte Carlo method (22). The conserved l-glycero-d-manno-heptopyranosyl trisaccharide (HepI–HepIII), depicted in red, is linked to the lipid A portion of the molecule (turquoise and gray) via a phosphorylated KDO residue (brown). The triheptosyl inner-core unit is substituted by a β-d-glucopyranose residue (Glc; green) at the O-4 position of HepI and by a phosphoethanolamine residue (PEtn; brown) at the O-6 position of HepII. In NTHi 375, 285, and 162 LPS, the Glc residue is substituted at O-6 by a phosphocholine residue (R1 = PCho; yellow). In NTHi strain 486 (R1 = H), HepII is substituted at the O-3 position by an α-d-glucopyranose which, in turn, is substituted by PCho at the O-6 position (R2 = PCho-6Glc). NTHi strain 375 can also carry a PEtn group on HepIII (R4 = H or PEtn). As shown in C, the NTHi strains used in this study can exhibit oligosaccharide chain extension from HepIII (R3) through sequential addition of sugar units (6, 7). LPS glycoforms containing β-d-glucopyranose, lactose, globotriose, and sialyllactose oligosaccharide chains have been identified (6, 7, 17, 23). The relative proportions of these glycoforms in the LPS from a particular strain depend on the expression of phase variable genes lic2A, lgtC, and lic3A (7, 24).

Fig. 4.

Negative ion CE-ESI-MS shows major glycoforms of LPS-OH extracted from NTHi strain 486. (A) Extracted mass spectrum for LPS-OH extracted from NTHi bacteria used to inoculate chinchillas. The mass spectrum is dominated by molecular peaks corresponding to doubly [(M-2H)^2–] and triply [(M-3H)^3–] charged ions. Triply charged ions at m/z 921 and 962 represent the lactose containing glycoform populations comprising four hexoses (Fig. 3A: R1 = H; R2 = phosphocholine residue-6Glc; R3 = lactose). The glycoform responsible for the ion at m/z 921 has a phosphate group at O-4 of the KDO residue, whereas that at m/z 962 carries a PPEtn group (Fig. 3A). Related doubly charged ions at m/z 1,382 and 1,444 are also observed. (B and C) Extracted mass spectra obtained from the CE-ESI-MS of exudate from the ear of a chinchilla 4 and 6 days, respectively, after inoculation with 50 WT organisms of the NTHi strain 486 described in A. In addition to ions corresponding to the Hex4 glycoforms observed in A, sialyllactose containing LPS species from the KDO-phosphate series of glycoforms are observed at m/z 764 [(M-4H)^4–], 1,018 [(M-3H)^3–], and 1,527 [(M-2H)^2–] in B. After 6 days, ions due to the sialylated glycoforms (e.g., m/z 1,018) are only just detectable above background (C).

Fig. 5.

Negative ion CE-ESI-MS with precursor ion monitoring of O-deacylated LPS extracted from NTHi strain 486. (A) Total ion electropherogram (m/z 600–1,600) obtained from the negative ion CE-ESI-MS of exudate from the ear of a chinchilla 5 days after inoculation with 50 WT organisms of the NTHi strain 486 described in Fig. 4A. (B) Precursor ion monitoring at m/z 220 of extracted LPS-OH spectrum shown in A. Triply charged ions corresponding to the lactose and sialyllactose containing Hex₄PEtn₂ glycoforms are observed at m/z 962 and 1,059. (C) Collisional activation (tandem MS) of ions at m/z 1,059. Major fragment ions at m/z 290 (Neu5Ac) are observed, confirming the presence of terminal sialic acid groups. (D) Precursor ion monitoring at m/z 290 (Neu5Ac) of extracted LPS-OH spectrum shown in A. Sialylated Hex4 glycoforms from both the KDO-P (m/z 1,018) and KDO-PPEtn (m/z 1,059) series are detected.