Role of Complement in Host Defense against Pneumococcal Otitis Media

Strategies to limit complement deposition on Streptococcus pneumoniae are established as virulence features for invasive disease, but their role in respiratory tract infection requires further analysis. We evaluated complement C3 protein deposition on discordant S. pneumoniae isolates of the same serotype (6A) and their capacity to cause nasopharyngeal (NP) colonization and experimental otitis media (EOM) in an animal model. We compared C3 binding to five 6A isolates from asymptomatic NP carriers with five 6A strains that caused invasive disease, and we observed less C3 (∼10-fold less fluorescence) binding to invasive isolates. We selected two high-level C3-binding carriage and two low-level C3-binding invasive 6A isolates for further study. In the EOM model, 11/12 (92%) ears challenged with a low-level C3-binding 6A strain became infected. Only 2/8 (25%) ears challenged with the discordant high-level C3-binding 6A isolate developed disease (P = 0.005). Results with the second discordant 6A isolate pair were comparable. Cobra venom factor (CoVF) treatment, which depletes C3 and consumes complement, restored virulence of the high-level C3-binding strain; 8/8 (100%) ears in CoVF-treated animals developed EOM compared to only 25% of ears in naïve animals (P = 0.007). These studies demonstrate the critical role for complement evasion in pneumococcal EOM. Colonization with carriage isolates that bound high levels of C3 caused EOM in fewer animals compared to low-level C3-binding invasive strains. Thus, limiting C3 deposition on the surface of S. pneumoniae correlates with increased incidence of EOM following NP colonization and barotrauma in the animal model.The pathogenesis of Streptococcus pneumoniae infection involves initial colonization of the nasopharynx, followed by its spreading to the middle ear, sinus, or lower respiratory tract and, in some cases, invasion of the bloodstream. To successfully cause disease, the pneumococcus has evolved a number of mechanisms to avert complement-mediated opsonization and phagocytosis. Pneumococci possess a broad variety of specialized surface proteins, some of which are adapted to interact with host defenses during colonization or dissemination in humans. Being a gram-positive bacterium, it is resistant to the bactericidal activity of complement (24) because its rigid cell wall prevents lysis by the membrane attack complex. The capsular polysaccharide is critical for resistance to complement deposition (32) and may also mask cell wall-associated complement from being recognized by the complement receptors on phagocytes (6). Additionally, select surface proteins can degrade native C3 proteins, thereby preventing or diminishing binding of C3b and iC3b to the bacterial surface, which are necessary components for opsonization (3). Furthermore, an important role for complement is suggested by the association of increased risk for invasive infections in individuals (or animal models) with deficiencies of complement proteins such as C2 and C3 and of complement receptors such as CR3 (2, 16). Type-specific antibody formation is an important host defense mechanism against infections caused by S. pneumoniae. However, the efficacy of opsonization of pneumococci by either immunoglobulin M (IgM) or IgG is related to their ability to enhance complement deposition on the bacterial surface, thus making complement essential for recovery from pneumococcal disease (6, 9).Colonization of mucosal surfaces is often the first step in the development of disease. Studies of S. pneumoniae support recent acquisition as the critical event preceding the development of pneumococcal otitis media. S. pneumoniae has evolved specific characteristics that are critical in dictating initial success for establishing colonization within a competitive niche of the mucosal surface of the nasopharynx. Often the success of an organism in establishing carriage depends on its ability to resist innate clearance mechanisms generated in the setting of polymicrobial stimulation. Lysenko and colleagues have demonstrated that complement and polymorphonuclear leukocytes are necessary host defenses for the elimination of S. pneumoniae from the nasopharynx in the presence of nontypeable Haemophilus influenzae (NTHi) (29).Prior work from our laboratory demonstrated that complement was an important host defense mechanism against protection of the middle ear from infection with NTHi (15). We hypothesized that complement would also be relevant for protection against S. pneumoniae respiratory tract infection (RTI). We evaluated the role of complement by comparing the capacities of four isolates of S. pneumoniae, all belonging to serotype 6A but differing in their abilities to bind complement to their surface to cause otitis media following nasopharyngeal (NP) colonization. Our goal was to determine whether evasion of complement deposition was an important virulence feature in the pathogenesis of RTI, using a model for experimental otitis media (EOM). The model requires initial NP colonization followed by ascension through the Eustachian tube after barotrauma for establishing middle ear infection.