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.Streptococcus pneumoniae is the most frequent cause of otitis media (OM) in children, accounting for 30% of the cases of acute OM and 5% of the cases of chronic OM with effusion (OME) (14). All S. pneumoniae isolates to date have been shown to produce neuraminidase (9). S. pneumoniae neuraminidase has been detected in 78% of culture-positive human middle ear effusions from patients with acute OM and in 96% of S. pneumoniae-positive middle ear effusions from patients with chronic OME (7). Neuraminidase is an enzyme that cleaves N-acetylneuraminic acid from mucin, glycolipids, glycoproteins, and oligosaccharides on host cell surfaces (18,20). Results of studies conducted in our laboratory indicate that during S. pneumoniae-induced OM in the chinchilla, terminal sialic acid residues are removed from the surface of the epithelium lining the lumen of the eustachian tube, presumably by neuraminidase, resulting in the exposure of GlcNAc ␤ 1-4 Gal, which is part of one of the S. pneumoniae eukaryotic receptors (10, 11). Additional studies conducted by our laboratory implicate neuraminidase as a virulence factor for S. pneumoniae (23,24). We found that the ability of a neuraminidase-deficient mutant to colonize and persist within the nasopharynx and middle ear was significantly impaired relative to the parent strain (21). These data indicate that neuraminidase plays an important role in the ability of S. pneumoniae to colonize and persist in the nasopharynx and induce OM. For this reason, we have chosen to investigate whether neuraminidase is a protective antigen and a potential protein-based vaccine candidate in the prevention of S. pneumoniae nasopharyngeal (NP) colonization and OM.Purification of native neuraminidase. Neuraminidase was purified from whole-cell lysates as previously described by Lock et al. (12). S. pneumoniae 6A (EF3114) was used for purification of native neuraminidase as well as intranasal inoculation in the chinchilla model of OM and has been described in detail previously (1,22). Fractions from a DEAESepharose column containing neuraminidase (identifi...

Versatility of pneumococcal surface proteins

“…In contrast, the nanA knockout is not attenuated in an intraperitoneal infection model (Berry & Paton, 2000). The importance of NanA for colonization has been underlined by its ability to afford protection against nasopharyngeal colonization and otitis media in chinchilla models (Long et al, 2004). Recently, two reports have demonstrated the precise function of NanA during pneumococcal colonization and pathogenesis.…”

The NanA Neuraminidase of Streptococcus pneumoniae Is Involved in Biofilm Formation

“…Pneumococcal neuraminidase activity also provides a source of carbohydrates for bacterial metabolism, cleaving sugars from the mucosal surface (8,23,61), but whether this significantly contributes to bacterial growth in vivo has not been clearly established. Several studies have suggested that nanA mutants colonize the rodent respiratory tract less efficiently than wild-type strains (31,40,52), and vaccination with purified NanA affords some protection against nasopharyngeal colonization and otitis media (29,30,53). However, the differences can be mouse strain and animal model dependent (6,13,22,23).…”

NanA, a Neuraminidase fromStreptococcus pneumoniae, Shows High Levels of Sequence Diversity, at Least in Part through Recombination withStreptococcus oralis

“…In patients with pneumococcal infection, a direct correlation exists between levels of N-acetylneuraminic acid in cerebrospinal fluid and development of coma and adverse outcome (31). Recently, immunization with NanA has also been demonstrated to afford protection against nasopharyngeal colonization and experimental otitis media (27).…”