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 Ϸ20% 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.ex vivo isolate ͉ phylogeny ͉ mass spectrometry C arried by up to 80% of humans, Haemophilus influenzae (Hi) is a common nasopharyngeal commensal. Capsule-deficient or nontypeable (NT) Hi can cause upper and lower respiratory tract infections, the most common being episodes of otitis media (OM) in young children. On average, children experience two or more episodes of acute OM by age 2 yr (1), making OM a major cause of physician visits (24 million per year in the U.S.) and of the profligate use of antibiotics in general practice. OM causes sequelae, including impaired hearing (Ϸ20% cases) and cognitive development (2). NTHi is also the most frequent pathogen recovered from the middle ear in children with recurrent OM (3). Although immunity against infection due to NTHi appears to develop after acute OM (4, 5), protection is strain specific, therefore permitting recurrent episodes due to distinct isolates.We have reported that all NTHi OM isolates have the potential to incorporate sialic acid [N-acetylneuraminic acid (Neu5Ac)] into their lipopolysaccharide (LPS), and that strains expressing this sugar are more resistant to the bactericidal activity of normal human serum in vitro (6-8). Although sialylation of LPS has been implicated in bacterial virulence (9), the contribution of sialylated LPS glycoforms of Hi has not been investigated in vivo.We have investigated the role of sialic acid as a virulence factor of NTHi in a well described chinchilla model of OM (10, 11). By comparing isogenic sialic acid-proficient and sialic acid-deficient strains, we show that sialylation of LPS is a major factor in...

Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

“…Moreover, prior work has clearly shown that NeuAc promotes biofilm formation and bacterial persistence in animal models of NTHi disease (4,18,43). Our previous studies did not show any biofilm phenotype associated with loss of PCho, at least in terms of initial attachment or numbers of viable bacteria found within biofilms (55).…”

“…LOS confers resistance to host killing (8,9,37) and is also the primary target of the Toll-like receptor 4 pathway that mediates protection against H. influenzae in the airways (47). It has been established that NTHi strains that express NeuAc-LOS forms comprise a greater proportion of biofilm communities than of planktonic cultures, and that mutations eliminating these forms decrease biofilm formation and bacterial persistence in animal models of OM (4,12,18,43). More recently, we showed that LOS purified from biofilms has decreased potency as an inflammatory agonist, which correlated with an increase in PCho ϩ LOS forms that were present within biofilms (55).…”

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

“…Previous studies of these four pathogens have linked LOS/LPS to adherence/virulence with full-length LOS/LPS for each of these organisms is absolutely required for virulence in animal models (40)(41)(42)(43). S. flexneri LPS O-antigen is a virulence factor whose chain length affects both serum resistance and the function of surface virulence factors; a role for LPS in cell invasion has been reported (11,(44)(45)(46).…”

Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c + cells through TLR4

“…Sialic acid is often found on pathogenic bacteria, such as C. jejuni, Haemophilus influenzae, Neisseria gonorrheae, Neisseria meningitidis, and Pasteurella multocida (27). The incorporation of host-derived α2,3-linked sialic acid into H. influenzae is a major virulence factor for experimental otitis (28). The presence of sialic acid on these bacteria is thought to mimic sialylated human glycans and thereby ease evasion from the host immune system.…”