Activation of Innate Immune Responses by Haemophilus influenzae Lipooligosaccharide

A Gram-negative pathogen Haemophilus influenzae has a truncated endotoxin known as lipooligosaccharide (LOS). Recent studies on H. influenzae LOS highlighted its structural and compositional implications for bacterial virulence; however, the role of LOS in the activation of innate and adaptive immunity is poorly understood. THP-1 monocytes were stimulated with either lipopolysaccharide (LPS) from Escherichia coli or LOS compounds derived from H. influenzae Eagan, Rd, and Rd lic1 lpsA strains. Cell surface expression of key antigen-presenting, costimulatory, and adhesion molecules, as well as gene expression of some cytokines and pattern recognition receptors, were studied. Eagan and Rd LOS had a lower capacity to induce the expression of ICAM-1, CD40, CD58, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) compared to LPS. In contrast, antigen-presenting (HLA-ABC or HLA-DR) and costimulatory (CD86) molecules and NOD2 were similarly upregulated in response to LOS and LPS. LOS from a mutant Rd strain (Rd lic1 lpsA) consistently induced higher expression of innate immune molecules than the wild-type LOS, suggesting the importance of phosphorylcholine and/or oligosaccharide extension in cellular responses to LOS. An LOS compound with a strong ability to upregulate antigen-presenting and costimulatory molecules combined with a low proinflammatory activity may be considered a vaccine candidate to immunize against H. influenzae.     

Association of IS1016 with the hia adhesin gene and biotypes V and I in invasive nontypeable Haemophilus influenzae

A subset of invasive nontypeable Haemophilus influenzae (NTHI) strains has evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. We examined IS1016-positive invasive NTHI isolates collected as part of Active Bacterial Core Surveillance within the Georgia Emerging Infections Program for the presence or absence of hmw1 and hmw2 (two related adhesin genes that are common in NTHI but absent in encapsulated H. influenzae) and hia (homologue of hsf, an encapsulated H. influenzae adhesin gene). Isolates were serotyped using slide agglutination, confirmed as NTHI strains using PCR capsule typing, and biotyped. Two hundred twenty-nine invasive NTHI isolates collected between August 1998 and December 2006 were screened for IS1016; 22/229 (9.6%) were positive. Nineteen of 201 previously identified IS1016-positive invasive NTHI isolates collected between January 1989 and July 1998 were also examined. Forty-one IS1016-positive and 56 randomly selected IS1016-negative invasive NTHI strains were examined. The hia adhesin was present in 39 of 41 (95%) IS1016-positive NTHI strains and 1 of 56 (1.8%) IS1016-negative NTHI strains tested; hmw (hmw1, hmw2, or both) was present in 50 of 56 (89%) IS1016-negative NTHI isolates but in only 5 of 41 (12%; all hmw2) IS1016-positive NTHI isolates. IS1016-positive NTHI strains were more often biotype V (P < 0.001) or biotype I (P = 0.04) than IS1016-negative NTHI strains, which were most often biotype II. Pulsed-field gel electrophoresis revealed the expected genetic diversity of NTHI with some clustering based on IS1016, hmw or hia, and biotypes. A significant association of IS1016 with biotypes V and I and the presence of hia adhesins was found among invasive NTHI. IS1016-positive NTHI strains may represent a unique subset of NTHI strains, with characteristics more closely resembling those of encapsulated H. influenzae.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota R4 (chemotype Rd2P-)

Rabbit polyclonal antibodies against the rough mutant lipopolysaccharide (LPS) of Salmonella minnesota R4 (chemotype Rd2P-) were serologically characterized by using R4 LPS, deacylated LPS, dephosphorylated LPS, and synthetic partial structures, including compounds comprising the core region of Rd2P- LPS bound to the beta 1-->6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or coupled to bovine serum albumin. By using a passive hemolysis assay and an enzyme immunoassay and absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core oligosaccharide (with or without the side chain 3-deoxy-D-manno-octulosonic acid [Kdo]) and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The phosphate-independent antibodies were directed against the core oligosaccharide, recognizing an epitope consisting of one terminal heptose linked to Kdo or to the reducing moiety of the alpha 2-->4-linked Kdo disaccharide. Antibodies requiring the presence of acyl residues and those reacting with a single heptose or Kdo residue were not detected.     

Potential of a Novel Protein, OMP26, from Nontypeable Haemophilus influenzae To Enhance Pulmonary Clearance in a Rat Model

A major outer membrane protein band of approximately 25 to 27 kDa is commonly observed in strains of Haemophilus influenzae. This study has investigated the potential of a 26-kDa protein (OMP26) from nontypeable H. influenzae (NTHI) as a vaccine candidate. OMP26 was used to immunize rats via intestinal Peyer’s patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain. Significant levels of anti-OMP26 were found in the serum and bronchoalveolar lavage from immunized rats, and isotypes of immunoglobulin G (IgG) were also measured in serum. Analysis of IgG isotypes present in serum following OMP26-immunization suggest that predominantly a T-helper 1-type response was induced. The OMP26 protein was amino-terminally sequenced and found to have no homology with the P5 of H. influenzae type b P5 or the fimbrin protein of NTHI, both can migrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis at similar molecular masses but OMP26 has 100% homology with a segment of the H. influenzae Rd genome. The results of this study suggest that OMP26 may be a suitable vaccine candidate against NTHI infection and warrants continued investigation and characterization.     

Elucidation of the monoclonal antibody 5G8-reactive, virulence-associated lipopolysaccharide epitope of Haemophilus influenzae and its role in bacterial resistance to complement-mediated killing

The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-alpha1,4-Gal-beta1,4-Glc-beta1,4-Glc-beta1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-alpha1,4-Gal-beta1,4-Glc-beta1,4-Glc-beta attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.     

Lex2B,a Phase-Variable Glycosyltransferase,Adds either a Glucose or a Galactose to Haemophilus influenzae Lipopolysaccharide

Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second β-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.Haemophilus influenzae is a gram-negative bacterium that is an obligate commensal in the human respiratory tract. This organism, however, has a propensity to spread contiguously, and the predominant unencapsulated (nontypeable H. influenzae [NTHi]) strains can cause diseases such as respiratory tract infections and otitis media (OM). Lipopolysaccharide (LPS), the major cell surface glycolipid, significantly influences host-microbe interactions and is a virulence determinant. From the well-conserved triheptose-containing inner core backbone, oligosaccharides (OS) comprising mainly hexose sugars and various nonsugar substituents extend, and these OS vary in composition between strains (31, 32, 36). The number of sugars and substituent groups comprising these OS can vary at a high frequency within individual strains, and the reversible loss and gain of epitopes is called phase variation (30). Phase variation of LPS in H. influenzae is mediated by switching in the expression of LPS-specific loci that contain tetranucleotide repeats (microsatellites) in the 5′ end of the reading frame (17, 19, 21, 38). These repeat tracts undergo mutation via DNA slippage during replication (25), leading to loss or gain of repeat units that place the reading frame in or out of frame with the translational initiation codon. Thus, depending on the number of tetranucleotide units in the tract, each phase-variable gene can be correctly translated (“on”) or not correctly translated (“off”). For any H. influenzae strain having multiple phase-variable loci, a population of cells can simultaneously generate a range of LPS glycoforms, allowing the organism to adapt to different microenvironments of the host and helping it escape from host immune responses.The lex2 locus, comprising lex2A and lex2B, was first identified as an H. influenzae phase-variable LPS biosynthetic locus in a serotype b strain, strain DL42 (21). A tract of multiple, tandem 5′-GCAA repeats is located in the 5′ end of the lex2A reading frame (21). Disruption of lex2B in strain DL42 led to a loss of reactivity of this strain with an LPS-specific monoclonal antibody (MAb), MAb 5G8 (21). Structural analysis of LPS isolated from mutant H. influenzae serotype b strains revealed that Lex2B functions as a glucosyltransferase that adds a second glucose in a β-(1-4) linkage to the first β-glucose linked to the first heptose of the LPS of H. influenzae (14) (Fig. ​(Fig.1).1). Both reading frames, lex2A and lex2B, are required for Lex2B activity (14). The lex2 locus permits further OS extensions that include a digalactoside that binds MAb 5G8 (13) (Fig. ​(Fig.1)1) and that play a role in resistance of the bacteria to the killing effect of serum (12).Open in a separate windowFIG. 1.Schematic representation of the structure of H. influenzae LPS, showing the relative position in biosynthesis that is influenced by the lex2 locus. Hex_II indicates the position of the sugar added by Lex2B (linkage indicated by an arrow) to GlcI. The example shown is the structure for a serotype b strain, RM7004 (29), where HexII is a glucose. The following molecules are present in the structure: Kdo, 2-keto-3-deoxyoctulosonic acid; Hep, l-glycero-d-manno-heptose; Hex, hexose (Glc or Gal); Glc, d-glucose; Gal, d-galactose; PEtn, phosphoethanolamine; P, phosphate; PC, phosphocholine.H. influenzae exhibits significant LPS structural variation both within and between strains. In addition to phase variation, factors that contribute to LPS heterogeneity are differences in the repertoire of LPS biosynthesis genes between strains and gene sequence polymorphisms that direct alternative transferase functions and additions to the LPS molecule. Depending on the allele present, the lic1D gene can direct addition of phosphocholine to one of several locations in the LPS (26), and the lpsA gene product can add either glucose or galactose in one of two alternative linkages to the same position in the LPS molecule (6).As part of our ongoing studies of the LPS structure and genetics of a set of 26 NTHi strains from OM patients (3), detailed analysis of the LPS from NTHi strains 432 and 1124 (40; unpublished data) has shown that the second hexose in the OS extension from HepI is a galactose rather than the glucose described previously at this position (14). We wanted to determine whether this sugar specificity was determined by lex2B alone, in a way analogous to that seen for the lpsA gene, or whether an alternative gene is involved. In the present study, we further characterize the lex2 locus of H. influenzae and show that the lex2B gene exhibits sequence polymorphism between strains that direct the attachment of either a β-glucose or a β-galactose as the second hexose in the OS extension from the proximal heptose of H. influenzae LPS. Lex2B is a glycosyltransferase that contributes to LPS structural heterogeneity through both its phase-variable expression and allelic polymorphisms that direct addition of alternative sugars.     

Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.     

Pasteurella multocida expresses two lipopolysaccharide glycoforms simultaneously, but only a single form is required for virulence: identification of two acceptor-specific heptosyl I transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo(1) residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.     

Binding specificity for four monoclonal antibodies recognizing terminal Galα1 4Gal residues in Haemophilus influenzae lipopolysaccharide

Four murine monoclonal antibodies (MAbs) reactive with the outer-core region of the lipopolysaccharide (LPS) fromHaemophilus influenzaewere generated after immunization with azide-killedH. influenzaeRM.7004 AH1-2 and their epitope specificities studied. The monoclonal antibodies: MAHI 6 (IgM), MAHI 5 (IgG2a), MAHI 8 (IgG3), and MAHI 11 (IgG2b) bound to synthetic glycoconjugates or glycolipids with terminal galabiosyl (Galα1 4Galβ1-) or globotriaosyl (Galα1 4Galβ1 1 4GLc) residues as evaluated in enzyme immunoassays (EIA). Glycoconjugates or glycolipids with internally placed galabiose elements were not active, indicating selectively of the MAbs for recognition of the epitope. Nine LPSs fromH. influenzaeinhibited the binding of the four MAbs. The presence of the galabiosyl disaccharide element in these nine LPSs was evidenced by the binding of125I-labeled Shiga toxin isolated from the bacteriumShigella dysenteriaetype 1, reported to have as receptor the Galα1 4Galβ disaccharide (Lindberget al., J Biol Chem, 1987, 262: 1779–85). Structural studies of theseH. influenzaeLPSs were also in accord with the presence of the galabiosyl disaccharide, in addition1H-NMR spectroscopy showed the presence of O-acetyl groups in the RM.7004 AH1-2 LPS. However, differential binding specificities of the MAbs to modified RM.7004 AH1-2 LPSs were observed. MAHI 6 and MAHI 11 bound equally well to LPS, polysaccharides obtained after mild acidic treatment, and dephosphorylated LPS samples as shown in inhibition EIA. In contrast, both dephosphorylated LPS samples and polysaccharides were poorer inhibitors of the binding of MAHI 5 fu1 and MAHI 8 to native RM.7004 AH1-2 LPS. Neither the de-O-acylated nor the de-O,N-acylated LPSs were effective inhibitors of any of the four MAbs. These results suggest that the MAbs recognition involves Galα1 4Gal and O-acetyl and other saccharide residue(s) from the oligosaccharide moiety of the LPS. The epitopes are also expressed and accessible to recognition in clinical isolates coming from different sources ofNeisseriaspp.,Haemophilusspp., andMoraxella catarrhalis, but not inBordetellaspp.,Aeromonasspp. orEnterobacteriaceaeas evaluated by whole-bacteria EIA and colony-dot-immunoblotting.     

Biofilm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus influenzae endotoxins

Nontypeable Haemophilus influenzae (NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation, PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated with PCho substitutions. We thus conclude that biofilm growth increases the proportion of PCho+ variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.     

A carcinoembryonic antigen-related cell adhesion molecule 1 homologue plays a pivotal role in nontypeable Haemophilus influenzae colonization of the chinchilla nasopharynx via the outer membrane protein P5-homologous adhesin

In vitro studies suggest an important role for CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) in infection by multiple gram-negative bacteria. However, in vivo evidence supporting this role is lacking, largely because the bacterial adhesins involved in this host-microbe association do not bind to murine-derived CEACAM1. One of several adhesins expressed by nontypeable Haemophilus influenzae (NTHI), the outer membrane protein P5-homologous adhesin (or P5), is essential for colonization of the chinchilla nasopharynx and infection of the middle ear. Here we reveal that NTHI P5 binds to the chinchilla homologue of CEACAM1 and that rabbit anti-human carcinoembryonic antigen blocks NTHI colonization of the chinchilla nasopharynx, providing the first demonstration of a role for CEACAM receptor binding by any bacterial pathogen in vivo.     

Diversity of Nontypeable Haemophilus influenzae Strains Colonizing Australian Aboriginal and Non-Aboriginal Children

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n = 81) and non-Aboriginal (n = 76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P < 0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P = 0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.     

Nontypeable Haemophilus influenzae Clearance by Alveolar Macrophages Is Impaired by Exposure to Cigarette Smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-α) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-α secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-α secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.The human respiratory tract is one of the largest body surfaces in contact with the environment and, therefore, is a main entry portal for microorganisms. In healthy humans, the lungs are sterile due to the combined actions of a repertoire of defense mechanisms. The components of lung innate immunity include mechanical barriers such as the mucociliary barrier, humoral elements present in the fluid in contact with the lung epithelium such as surfactants, complement, antimicrobial peptides, lysozyme, and lactoferrin, and resident innate immunity cells such as alveolar macrophages and dendritic cells (32, 37). Alveolar macrophages are professional phagocytes and antigen-presenting cells which patrol the lungs as sentinels and are endowed with, among other things, a collection of pattern recognition receptors used to recognize microorganisms containing pathogen-associated molecular patterns. As professional phagocytes, alveolar macrophages recognize, ingest, and process foreign material using a phagolysosomal pathway and thus play an essential role in the clearance of infections (18).Cigarette smoke is the main risk factor for the development of lung cancer, chronic obstructive pulmonary disease (COPD), and respiratory infections (26). In this context, the so-called “British hypothesis” states that recurrent bronchial infections were the reason, at least partially, that some smokers developed progressive airway obstruction and others did not (12, 13). Exposure to cigarette smoke markedly alters lung immunity by disruption of the mucociliary function, mucus hypersecretion, and disturbance of the mucosal integrity (31). Cigarette smoke also causes oxidative stress which triggers local lung inflammation by activation of epithelial cells, alveolar macrophages, neutrophils, and T lymphocytes (2). These cells secrete inflammatory cytokines, proteases, and reactive oxygen species, causing necrosis, tissue damage, and further amplification of the inflammatory response with enhanced recruitment of neutrophils into the lung. Tissue damage promotes the release of inflammatory mediators and inhibits lung tissue repair functions, further increasing the tissue damage in the lungs of smokers (35, 38, 39). It is generally accepted, although it has not been formally proven, that these alterations could allow access of microorganisms to the otherwise sterile lungs, thereby leading to microbial colonization (28-30). Supporting this hypothesis, mice exposed to cigarette smoke were impaired in the ability to clear a Pseudomonas aeruginosa infection (10). However, there is currently limited information concerning the effect of cigarette smoke at the molecular and cellular levels on the interaction between pathogens and alveolar macrophages.Glucocorticoids are drugs that are widely used to control many inflammatory and immune diseases, including respiratory diseases. Moreover, adjunctive glucocorticoid therapy is currently being used against a variety of bacterial infections, including otitis media, and COPD (7, 21). However, despite their importance in suppressing inflammatory responses, little is known about the effects of glucocorticoids in host defense against pathogens.Nontypeable Haemophilus influenzae (NTHI) is a frequent gram-negative asymptomatic colonizer of the upper respiratory tract in healthy humans, but it is also an opportunistic bacterial pathogen. NTHI causes invasive diseases such as meningitis and acute respiratory infections such as otitis media with effusion, sinusitis, pneumonia, and bronchitis (24). Moreover, NTHI is the pathogen isolated most frequently from lower respiratory tract secretions from patients suffering from chronic respiratory diseases such as COPD and chronic bronchitis (30). Lipooligosaccharide (LOS) is the main glycolipid on the NTHI cell surface and comprises a membrane-anchoring lipid A molecule linked to oligosaccharide chains that extend from the bacterial cell surface (27). Phosphocholine (PCho) is a substituent frequently present in NTHI LOS chain extensions (36). This modification has been shown to be a virulence factor that is involved in NTHI adhesion and invasion of the respiratory epithelium and hence promotes pathogen persistence on the mucosal surface of the respiratory tract (33, 34).The importance of NTHI as a respiratory pathogen has been extensively demonstrated, and alveolar macrophages play an essential role in the clearance of bacterial infections. However, little is known about the interaction between NTHI and alveolar macrophages and about the influence of PCho on this interaction. It is tempting to speculate that NTHI might be able to escape alveolar macrophage-mediated killing and that PCho could play an important role in this process. In addition, given the association between cigarette smoke and respiratory infections caused by NTHI, we hypothesized that cigarette smoke could modify the characteristics of the interaction between NTHI and alveolar macrophages. In the present study, we investigated the features of the interaction between NTHI and alveolar macrophages. Furthermore, we analyzed the impact of cigarette smoke on the ability of alveolar macrophages to engulf and process this respiratory pathogen and whether glucocorticoids affect this interaction.     

Cloning and molecular analysis of theIsi1(rfaF) gene ofNeisseria meningitidiswhich encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells

Neisseria meningitidis, but notHaemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. TheIsi1gene, which had been shown to be involved in LPS biosynthesis ofNeisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology withIsi1ofN. gonorrhoeaeand therfaFgene ofSalmonella typhimuriumwhich encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the clonedIsi1gene, as a functionalrfaFhomologue, was confirmed by the complementation of aS. typhimurium rfaFmutant using a P22 phage sensitivity test. AnIsi1mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type,Isi1, andgalEstrains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galEmutant) or the majority (Isimutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity.     

Peroxiredoxin-Glutaredoxin and Catalase Promote Resistance of Nontypeable Haemophilus influenzae 86-028NP to Oxidants and Survival within Neutrophil Extracellular Traps

Nontypeable Haemophilus influenzae (NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded by hktE) and a bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX) in resistance of NTHI to oxidants and persistence in vivo. Isogenic NTHI strain 86-028NP mutants lacking hktE and pdgX had increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed that pdgX and hktE were important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilms in vivo. Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.     

Molecular biology of haemophilus influenzae IgA1 proteases

IgA1 proteases of two distinct specificities were demonstrated among 95 isolates of Haemophilus influenzae and nine isolates of H. aegyptius. The two enzymes cleaved two different peptide bonds in the hinge region of the α chain of IgA1: a prolyl-seryl bond located at position 231–232 (type A cleavage) and a prolyl-threonyl peptide bond between residues 235 and 236 (type B cleavage). Each strain of H. influenzae produced either one or both of these types of enzymes, whereas all H. aegyptius strains produced type A enzyme only. The application of enzyme-neutralizing antibodies to the study of IgA1 proteases produced by the 104 strains of H. influenzae and H. aegyptius revealed at least 15 different types of protease activities based on inhibition patterns in nine selected antibody preparations. The types of IgA1 proteases closely correlated with the serotype of encapsulated strains of H. influenzae. The study suggests that H. influenzae strains produce at least two serologically different IgA1 proteases with distinct or identical enzymatic activities.     

Antigenic diversity of Haemophilus somnus lipooligosaccharide: phase-variable accessibility of the phosphorylcholine epitope

The lipooligosaccharide (LOS) of Haemophilus somnus undergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in H. somnus has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from H. somnus strains and phase variants. Clinical isolates of H. somnus exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glucose residues in this region. Our results indicate that outer core epitopes of H. somnus LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.     

Therapeutic Transcutaneous Immunization with a Band-Aid Vaccine Resolves Experimental Otitis Media

Transcutaneous immunization (TCI) is a noninvasive strategy to induce protective immune responses. We describe TCI with a band-aid vaccine placed on the postauricular skin to exploit the unique organization of the stratum corneum and to promote the development of immune responses to resolve active experimental otitis media due to nontypeable Haemophilus influenzae (NTHI). This therapeutic immunization strategy induced significantly earlier resolution of middle ear fluid and rapid eradication of both planktonic and mucosal biofilm-resident NTHI within 7 days after receipt of the first immunizing band-aid vaccine. Efficacy was ascribed to the homing of immunogen-bearing cutaneous dendritic cells to the nasal-associated lymphoid tissue, induction of polyfunctional CD4⁺ T cells, and the presence of immunogen-specific IgM and IgG within the middle ear. TCI using band-aid vaccines could expand the use of traditional parenteral preventative vaccines to include treatment of active otitis media, in addition to other diseases of the respiratory tract due to NTHI.