Nontypeable Haemophilus Influenzae Lipooligosaccharide Research Articles

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

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.

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

Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

Serum IgM and C-Reactive Protein Binding to Phosphorylcholine of Nontypeable Haemophilus influenzae Increases Complement-Mediated Killing.

Nontypeable Haemophilus influenzae (NTHi) colonizes the human upper respiratory tract without causing disease symptoms, but it is also a major cause of upper and lower respiratory tract infections in children and elderly, respectively. NTHi synthesizes various molecules to decorate its lipooligosaccharide (LOS), which modulates the level of virulence. The presence of phosphorylcholine (PCho) on NTHi LOS increases adhesion to epithelial cells, which is an advantage for the bacterium enabling nasopharyngeal colonization. However, when PCho is incorporated on the LOS of NTHi, it is recognized by the acute-phase C-reactive protein (CRP) and PCho-specific antibodies, both potent initiators of the classical pathway of complement activation. We determined the presence of PCho and binding of IgG and IgM to the bacterial surface for 319 NTHi strains collected from the nasopharynx/oropharynx, middle ear, and lower respiratory tract. PCho detection was higher for NTHi strains collected from the nasopharynx/oropharynx, which was associated with increased binding of IgM and IgG to the bacterial surface. Binding of CRP and IgM to the bacterial surface of PChohigh NTHi strains increased complement-mediated killing, which was largely dependent on PCho-specific IgM. The levels of PCho-specific IgM varied in sera from 12 healthy individuals, and higher PCho-specific IgM levels were associated with increased complement-mediated killing of a PChohigh NTHi strain. In conclusion, incorporation of PCho on the LOS of NTHi marks the bacterium for binding of CRP and IgM, resulting in complement-mediated killing. Therefore, having a lower PCho might be beneficial in situations where sufficient PCho-specific antibodies and complement are present.

Nontypeable Haemophilus influenzae Invasive Blood Isolates Are Mainly Phosphorylcholine Negative and Show Decreased Complement-Mediated Killing That Is Associated with Lower Binding of IgM and CRP in Comparison to Colonizing Isolates from the Oropharynx

Nontypeable Haemophilus influenzae (NTHi) bacteria express various molecules that contribute to their virulence. The presence of phosphocholine (PCho) on NTHi lipooligosaccharide increases adhesion to epithelial cells and is an advantage for the bacterium, enabling nasopharyngeal colonization, as measured in humans and animal models. However, when PCho is expressed on the lipooligosaccharide, it is also recognized by the acute-phase protein C-reactive protein (CRP) and PCho-specific antibodies, both of which are potent initiators of the classical pathway of complement activation. In this study, we show that blood isolates, which are exposed to CRP and PCho-specific antibodies in the bloodstream, have a higher survival in serum than oropharyngeal isolates, which was associated with a decreased presence of PCho. PCholow strains showed decreased IgM, CRP, and complement C3 deposition, which was associated with increased survival in human serum. Consistent with the case for the PCholow strains, removal of PCho expression by licA gene deletion decreased IgM, CRP, and complement C3 deposition, which increased survival in human serum. Complement-mediated killing of PChohigh strains was mainly dependent on binding of IgM to the bacterial surface. These data support the hypothesis that a PCholow phenotype was selected in blood during invasive disease, which increased resistance to serum killing, mainly due to lowered IgM and CRP binding to the bacterial surface.

Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia.

Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells.

Differential expression of cytokine genes and iNOS induced by nonviable nontypeable Haemophilus influenzae or its LOS mutants during acute otitis media in the rat

We have previously demonstrated that the disruptions of nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) htrB and rfaD genes may play a role in the pathogenesis of otitis media (OM). The purpose of this study was to determine whether NTHi LOS gene disruptions influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of acute OM. At 3, 6, 12, 24, 48 and 72 h after transbullar inoculation with nonviable NTHi, expression of genes for the cytokines and chemolines; tumor necrosis factor alpha (TNF-alpha), interleukin-lbeta (IL-1beta), and IL-6, IL-1alpha, IL-8, IL-10, and inducible nitric oxide synthase (iNOS) were quantitated by real-time PCR. Enzyme-linked immunosorbent assay was performed to confirm the gene expression data as determined by real-time PCR. The middle ear inflammatory responses were also evaluated. The NTHi 2019 parent and its isogenic LOS htrB (B29) and rfaD (DK-1) mutant strains induced a significant up-regulation in gene expression for the cytokines examined compared to the sham-inoculated controls at 3, 6 and 12 h post-inoculation (P<0.05 in all cases). However, the NTHi 2019 cohort demonstrated a significant increase in gene expression for TNF-alpha (up to 6 h), IL-1alpha and IL-8 (up to 24 h), IL-1beta and IL-6 (up to 48 h), and IL-10 and iNOS (up to 72 h) relative to the animals inoculated with NTHi B29 (P<0.05, in all cases), Moreover, the concentrations of inflammatory cells in the middle ear lavage fluid samples from the NTHi 2019 cohort were 2.8-5.3-fold higher than those of the B29 cohort. There were no significant differences in mRNA expression of the cytokines between the NTHi 2019 and the DK-1-treated groups. Data from this study indicate that the disruption of the NTHi htrB gene may impact the temporal mRNA expression of inflammatory mediators and inflammation within the middle ear.

Development of peptide mimotopes of lipooligosaccharide from nontypeable Haemophilus influenzae as vaccine candidates.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media in children and lower respiratory tract diseases in adults. So far there is no effective vaccine against NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective Ag. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent Ag through the use of peptides that mimic the LOS by screening a phage-display peptide library with a rabbit Ab specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified as NMMRFTSQPPNN, NMMNYIMDPRTH, NMMKYISPPIFL, and NMMRFTELSTPS. Three of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS Ab and also a mouse bactericidal monoclonal anti-LOS Ab in vitro, and elicited specific serum anti-LOS Abs in rabbits (27- to 81-fold) after conjugation with keyhole limpet hemocyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was preabsorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi.

Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor.

Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB:ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB:ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.

The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274.

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue.