Underlying Glycans Determine the Ability of Sialylated Lipooligosaccharide To Protect Nontypeable Haemophilus influenzae from Serum IgM and Complement

Abstract

Nontypeable Haemophilus influenzae (NTHi) efficiently colonizes the human nasopharynx asymptomatically but also causes respiratory mucosal infections, including otitis media, sinusitis, and bronchitis. The lipooligosaccharide (LOS) on the cell surface of NTHi displays complex glycans that mimic host structures, allowing it to evade immune recognition. However, LOS glycans are also targets of host adaptive and innate responses. To aid in evasion of these responses, LOS structures exhibit interstrain heterogeneity and are also subject to phase variation, the random on/off switching of gene expression, generating intrastrain population diversity. Specific LOS modifications, including terminal sialylation of the LOS, which exploits host-derived sialic acid (Neu5Ac), can also block recognition of NTHi by bactericidal IgM and complement by mechanisms that are not fully understood. We investigated the LOS sialic acid-mediated resistance of NTHi to antibody-directed killing by serum complement. We identified specific LOS structures extending from heptose III that are targets for binding by naturally occurring bactericidal IgM in serum and are protected by sialylation of the LOS. Phase-variable galactosyltransferases encoded by lic2A and lgtC each add a galactose epitope bound by IgM that results in antibody-dependent killing via the classical pathway of complement. NTHi’s survival can be influenced by the expression of phase-variable structures on the LOS that may also depend on environmental conditions, such as the availability of free sialic acid. Identification of surface structures on NTHi representing potential targets for antibody-based therapies as alternatives to antibiotic treatment would thus be valuable for this medically important pathogen.

Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae

“…Our data present a unique role of Kdn contributing to the reduction of serum and whole blood survival through the increased deposition of complement C3 and IgM antibody. Kdn incorporation could not only present new targets but also potentially uncover the nonself (bacterial) surface epitopes, otherwise masked by Neu5Ac capping, to immune responders, thus corroborating other antimicrobial mechanisms that target non-Neu5Ac structures (23,91). The presence of Kdn reduces the engagement of Siglec-9, an inhibitory Siglec widely expressed on circulating leukocytes in humans ( 124) that Neu5Accoated microbes exploit to dampen the immune activation (125).…”

“…Unlike Neu5Ac, Kdn does not protect against serum and whole blood killing. Incorporation of Neu5Ac on LOS allows NTHi to resist complement-mediated serum killing (88)(89)(90)(91). Since Kdn can be incorporated into LOS, we asked whether its presence affected NTHi survival in human sera.…”

“…Human serum bactericidal effects against NTHi are mainly attributed to complement-mediated and antibody-dependent cell cytotoxicity (81,89,95,96). The presence of Sia on NTHi LOS significantly reduces IgM binding to promote resistance against antibody-mediated immune clearance (91,97). In contrast to IgG, IgM binding is correlated with complement-mediated NTHi killing (98).…”

Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands

“…Bacterial mechanisms that result in evasion of IgM binding to the bacterial surface are diverse. For instance, incorporation of sialic acids decreases the binding of IgM to the bacterial surface (9), possibly due to covering galactose residues extending on heptose III of the LOS structure (12). Decreased incorporation of phosphorylcholine into the LOS of NTHi decreased the binding of IgM as well (10), which was consistent with having a lower phosphorylcholine expression and IgM binding to the surface of NTHi strains collected from the blood of a patient with bacteremia (4).…”

A Bacterial Epigenetic Switch in Non-typeable Haemophilus influenzae Modifies Host Immune Response During Otitis Media

“…OMPs facilitate bacterial adherence to mucosal surfaces during both colonization and infection, and can induce innate immune response (Berenson et al, 2005;Punturieri et al, 2006). LOS has been shown to be both immunostimulatory as well as immunoevasive, dependent on the composition and structure of the molecule (Hong et al, 2009;Juneau et al, 2011;Jackson et al, 2019;Ng et al, 2019). Regulation of the genes involved in each of these processes can have substantial impact on pathogenesis of this human pathobiont.…”