Hua Hua Tong

Evaluation of the virulence of a Streptococcus pneumoniae neuraminidase-deficient mutant in nasopharyngeal colonization and development of otitis media in the chinchilla model.

ABSTRACT Considerable evidence has implicated Streptococcus pneumoniae neuraminidase in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, an S. pneumoniae neuraminidase-deficient mutant, ΔNA1, has been constructed by insertion-duplication mutagenesis of the nanA gene of S. pneumoniae strain D39. The relative ability of ΔNA1 and the D39 parent strain to colonize the nasopharynx and to induce OM subsequent to intranasal inoculation and to survive in the middle ear cleft after direct challenge of the middle ear were evaluated in the chinchilla model. Nasopharyngeal colonization data indicate a significant difference in the ability of the ΔNA1 mutant to colonize as well as to persist in the nasopharynx. The neuraminidase-deficient mutant was eliminated from the nasopharynx 2 weeks earlier than the D39 parent strain. Both the parent and the mutant exhibited similar virulence levels and kinetics during the first week after direct inoculation of the middle ear. The ΔNA1 neuraminidase-deficient mutant, however, was then completely eliminated from the middle ear by day 10 postchallenge, 11 days before the D39 parent strain. Data from this study indicate that products of thenanA gene have an impact on the ability of S. pneumoniae to colonize and persist in the nasopharynx as well as the middle ear.

Evaluation of Phase Variation of Nontypeable Haemophilus influenzae Lipooligosaccharide during Nasopharyngeal Colonization and Development of Otitis Media in the Chinchilla Model

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) has four loci, lic-1 to lic-3 and lgtC, that generate phase-variable lipooligosaccharide (LOS) structures.lic-1, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of H. influenzae to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of lic-1 tolic-3 and lgtC of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP− to the ChoP+ phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP+ phenotype demonstrated a significantly higher level of NTHI 2019 per milliliter of nasal lavage fluid than chinchillas colonized with predominantly the ChoP− variant (P < 0.05). The concentration of cells with the ChoP+ phenotype in the middle ear was 3 log units higher than that of cells with the ChoP− variant (P < 0.01). There was a statistically significant association between ChoP+ expression in the nasal lavage and the development of OM with culture-positive middle ear fluids in this model. These data suggest that expression of the ChoP+phenotype promotes enhanced nasopharyngeal colonization and development of OM.

Effect of Neuraminidase on Receptor-mediated Adherence of Streptococcus pneumoniae to Chinchilla Tracheal Epithelium

The trachea whole organ perfusion technique was used to study the effect of the disruption of the Streptococcus pneumoniae neuraminidase nanA gene on bacterial adherence and alteration of the carbohydrate surface structures of respiratory epithelial cells. Six different lectin probes were used to examine alterations of the cell surface carbohydrates in chinchilla tracheal epithelium incubated in vitro with S. pneumoniae j NA1, a neuraminidase-deficient mutant, or its D39 parent strain. The labeling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated WGA was significantly increased in the luminal surface of the trachea in the D39-incubated cohort compared with the uninfected control, which indicated that GlcNAc and D-galactose residues were exposed. Concurrently, decreased labeling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the tracheal epithelium subsequent to incubation with D39. The j NA1 neuraminidase-deficient mutant, however, did not induce any significant changes in the lectin labeling patterns, which were comparable to those of the control cohort. Moreover, adherence data expressed as colony-forming units (CFU) of S. pneumoniae per millimeter of trachea indicated a significant decline in the ability of j NA1 to adhere in vitro . We propose that products of the nanA gene have a significant impact on changes in the carbohydrate moieties in the tracheal epithelium, and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

Effect of Influenza A Virus Infection on Nasopharyngeal Colonization and Otitis Media Induced by Transparent or Opaque Phenotype Variants of Streptococcus pneumoniae in the Chinchilla Model

ABSTRACT Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal disease. Additionally, the synergistic effects of influenza A virus and S. pneumoniae in the development of otitis media (OM) have been reported. This study examined the ability of opaque or transparent S. pneumoniae from the same strain in combination with an antecedent influenza A virus infection to colonize the nasopharynx and invade the middle ear in the chinchilla model. Our data indicated that there was no significant difference in the level of nasopharyngeal colonization and induction of OM between the opaque and transparent variants unless there was a prior challenge with influenza A virus. Subsequent to influenza A virus infection, there was a significant difference between the variants in the ability to colonize and persist in the nasopharynx and middle ear. The concentrations of the opaque variant in nasopharyngeal-lavage samples and middle-ear fluid remained consistently higher than those of the transparent variant for 10 days postinoculation. Data from this study indicate that the effects of influenza A virus on the pathogenesis of experimental S. pneumoniae-induced OM differ depending on the opacity phenotype involved.

Immunization with native or recombinant Streptococcus pneumoniae neuraminidase affords protection in the chinchilla otitis media model.

ABSTRACT Streptococcus pneumoniae neuraminidase has been implicated as a virulence factor in the pathogenesis of pneumococcal otitis media. In this study, native neuraminidase was partially purified from cultures of S. pneumoniae by serial chromatography with DEAE-Sepharose and Sephacryl S-200. Recombinant neuraminidase, a 3,038-bp fragment of the neuraminidase A (nanA) gene, was cloned into the pET-28b vector and then expressed at high levels in Escherichia coli. Chinchillas were immunized subcutaneously with either the gel-purified native or recombinant neuraminidase, and all responded with elevated titers of antineuraminidase antibody in serum. Immunization with neuraminidase resulted in a significant reduction in nasopharyngeal colonization as well as in the incidence of otitis media with effusion. These data demonstrate for the first time that neuraminidase affords protection against S. pneumoniae nasopharyngeal colonization and experimental otitis media.

Effect of Adenovirus Type 1 and Influenza a Virus on Streptococcus Pneumoniae Nasopharyngeal Colonization and Otitis Media in the Chinchilla

Considerable evidence has implicated respiratory tract virus potentiation of bacterial adherence, colonization, and superinfection as a significant factor contributing to the pathogenesis of otitis media (OM). Influenza A and B viruses, adenovirus, and respiratory syncytial virus are the primary respiratory tract viruses associated with this disease. Investigations have established a dramatic increase in the development of experimental OM in chinchillas co-inoculated with influenza A virus and Streptococcus pneumoniae (Spn). The mechanism underlying this phenomenon was suggested to involve, in part, viral compromise of eustachian tube mucosal integrity and function. This study was designed to assess and compare the effect of adenovirus and influenza A virus infection on adherence, the kinetics of colonization, and invasion of the middle ear by Spn in the chinchilla model of OM. Cohorts were inoculated intranasally with adenovirus type 1 or influenza A virus, and then inoculated intranasally 7 days later with Spn 6A. All cohorts were observed over a 14-day period after challenge with Spn, and the incidence and severity of OM were assessed by several methods, including culture of the nasopharynx and middle ear effusions. The data indicated that influenza A virus promotes a significant increase in nasopharyngeal colonization by Spn, an increased incidence and severity of OM, and a sustained presence of Spn in the effusions. Adenovirus infection, however, did not enhance colonization by Spn or result in an increased incidence or severity of OM.

Differential Expression of Cytokine Genes and Inducible Nitric Oxide Synthase Induced by Opacity Phenotype Variants of Streptococcus pneumoniae during Acute Otitis Media in the Rat

ABSTRACT Phase variation in the colonial opacity phenotype of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal otitis media (OM). The purpose of this study was to determine whether S. pneumoniae opacity variants influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of OM. Both the opaque and transparent phenotype variants induced a significant up-regulation in gene expression for interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) compared to saline sham-inoculated controls at both 4 and 24 h postinoculation (P < 0.05 in all cases). Furthermore, whereas a significant difference in gene expression was evident for only IL-6 (greater following challenge with the opaque variant) and IL-1β (greater following challenge with the transparent variant) at 4 h, by 24 h the opaque variant cohort demonstrated a significant increase in gene expression for IL-1α, IL-1β, IL-6, IL-10, and iNOS relative to animals inoculated with the transparent phenotype variant (P < 0.05 in all cases). Enzyme-linked immunosorbent assay results confirmed the gene expression data as determined by real-time PCR. Moreover, the concentrations of the opaque variant in the middle ear lavage fluid were a full log higher than those of the transparent variant. The aforementioned results indicate that the opaque phenotype variant is more efficient at survival and multiplication within the middle ear space, resulting in the accumulation of more inflammatory cells and the enhanced expression and production of inflammatory mediators. However, when the data were normalized to account for differences in middle ear bacterial titers, it became apparent that the transparent variant of S. pneumoniae is a more potent inducer of inflammation, triggering the accumulation of more inflammatory cells and substantially greater fold increases in the expression and production of inflammatory mediators. Data from this study indicate that S. pneumoniae opacity variants influence the temporal mRNA expression of inflammatory mediators within the middle ear.

Immunization with recombinant Streptococcus pneumoniae neuraminidase NanA protects chinchillas against nasopharyngeal colonization

ABSTRACT Immunization with recombinant S. pneumoniae neuraminidase NanA (rNanA) resulted in a significant reduction in pneumococcal colonization in the chinchilla model. The bacteria were eliminated from the nasopharynx 1 week earlier than that from the control cohort. Our data suggest that rNanA affords protection against pneumococcal nasopharyngeal colonization.

Comparison of alteration of cell surface carbohydrates of the chinchilla tubotympanum and colonial opacity phenotype of Streptococcus pneumoniae during experimental pneumococcal otitis media with or without an antecedent influenza A virus infection

ABSTRACT Experimental and clinical studies suggest that influenza A virus promotes Streptococcus pneumoniae-induced otitis media; however, the mechanism underlying this synergistic interaction has not been completely defined. In this study, glycoconjugate expression patterns were evaluated on the cell surface in the chinchilla eustachian tube (ET) lumen of a cohort challenged intranasally (i.n.) with S. pneumoniae type 6A, which is predominantly transparent and a cohort with an antecedent influenza A virus infection, followed by i.n. inoculation with S. pneumoniae. The labeling patterns obtained with six lectin probes revealed that the binding of Bandeiraea simplicifolia lectin II, succinylated wheat germ agglutinin, and peanut agglutinin were significantly increased in the lumenal surface of the ET in the cohort infected with both pathogens compared to the cohort inoculated with only S. pneumoniae, which indicated that N-acetylglucosamine (GlcNAc) and d-galactose residues were exposed. A significant decreased labeling with Sambucus nigra agglutinin in the combined influenza A virus and pneumococcus infection cohort suggested that there were few sialic acid residues remaining in the ET epithelium. In addition, the colonial opacity of S. pneumoniae during the disease course was examined. The opaque phenotype was predominant among the pneumococcus isolates from the middle-ear fluid in the cohort infected with the both pathogens. Together, these data suggest that the synergic effect of influenza A virus and S. pneumoniae on the changes of the carbohydrate moieties in the ET epithelium and that the selection of the opaque variant may facilitate the pneumococcal invasion of the middle ear.

Enhanced Susceptibility to Acute Pneumococcal Otitis Media in Mice Deficient in Complement C1qa, Factor B, and Factor B/C2

ABSTRACT To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2−/−), C1qa (C1qa−/−), and factor B (Bf−/−). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2−/−, Bf−/−, and C1qa−/− mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2−/− mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2−/−, Bf−/−, and C1qa−/− mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2−/− mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa−/− and Bf−/− mice, and least for Bf/C2−/− mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.