Virulence of Pneumococcal Proteins on the Inner Ear

Objective  To investigate the effects of the virulence characteristics of specific pneumococcal proteins on the inner ear.

Main Outcome Measures  A histologic comparison of inflammatory cell infiltration and pathologic changes in the round window membrane and inner ear.

Results  Most of the animals inoculated with high-dose pneumolysin or wild-type bacteria showed severe pathologic changes of the inner ears. The inner ears of most animals inoculated with surface protein A or surface antigen A–deficient bacteria appeared normal.

Conclusions  Pneumococcal surface protein A and pneumococcal surface antigen A are 2 important virulence factors in inner ear damage secondary to pneumococcal otitis media. Mutation of these virulence factors results in less inner ear damage.

Otitis media (OM) is one of the most common childhood diseases. Teele et al¹ reported that 80% of children have 1 episode of OM by 3 years of age. In a study of antibiotic use and infections in infants and young children,² OM was the most common indication and was responsible for 67.3% of antibiotics prescribed. The emergence of antibiotic-resistant bacterial strains has become a major health concern. Streptococcus pneumoniae is one of the important pathogens in acute OM, and its local rates of resistance have been reported to be from 50% to 60%.³

Antibiotic-resistant microorganisms have led to an increase in suppurative complications in OM.⁴ Redaelli de Zinis et al⁵ found middle-ear disease the most predisposing factor to sensorineural hearing loss and duration of disease to be related to its severity. Bacterial resistance to antibiotics has led to the development of alternatives such as the polyvalent vaccine. Although the Pneumovax 23 valent polysaccharide vaccine (Merck & Co, Whitehouse Station, New Jersey) is immunogenic and protective in most adults and children older than 5 years, it is not effective in children younger than 2 years,⁶ and conjugate vaccines have only limited serotype protection. This led to consideration of several pneumococcal proteins as alternative vaccine candidates; among these are pneumococcal surface protein A (PspA), pneumococcal surface antigen A (PsaA), and pneumolysin (Ply).

In a previous study, we demonstrated that pneumococci deficient in PspA (PspA⁻) and PsaA proteins were less likely to pass from the middle ear through the round window membrane and into the inner ear than the wild-type or Ply-deficient (Ply⁻) mutant bacterial strain (hereinafter, mutant).⁷ In this study, we compare inner-ear damage following high-dose and low-dose inoculations of wild-type mutant bacterial strains of these proteins into the middle ear.

Sixty-three young chinchillas weighing 250 to 350 g were included in this study. The care and use of these animals were approved by the Institutional Animal Care and Use Committee of the University of Minnesota, Minneapolis. All animals were anesthetized prior to bacterial inoculation with a combination of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (10 mg/kg). They were inoculated bilaterally, with 0.5 mL of a high dose (10⁵ to 10⁷ colony-forming units [CFUs]) or low dose (10³ CFUs) of S pneumoniae serotype 2 strain 39 (NCTC 7466) or its isogenic mutant deficient in PspA, PsaA⁻ mutant, that was derived by insertional inactivation mutagenesis of the PspA gene that was an insertional duplication derivate of the 7455 parent strain or the Ply⁻ mutant that had an insertionally inactivated Ply gene. Bacteria were grown in Todd-Hewitt broth (BD Diagnostics, Sparks, Maryland) containing yeast extract and plated on sheep blood agar. Erythromycin was added to plates for growth of the mutants, and bacteria were stored in glycerin freezing solution at −80°C. Bacteria were grown until they were in log phase, and their optical densities were measured. Concentrations were estimated, based on the specific strain, and diluted to the desired concentration. Ten-fold dilutions were plated, and viable cells were counted to confirm the actual concentration.

Two days after inoculation, animals were killed by overdose of sodium pentobarbital. The bullae were removed, and the middle-ear effusions grossly classified as clear or cloudy, yellow or white, and thick or thin. Cochleas were then gently perfused via the apex and oval window with glutaraldehyde, 2%, in 0.2M of phosphate buffer (pH, 7.4) for 2 hours. Samples were decalcified in ethylenediaminetetraacetic acid, 10%, for 3 days, washed in phosphate buffer, postfixed in osmium tetroxide, 1%, in 0.2M of phosphate buffer for 1 hour, and again washed with buffer. Following fixation, samples were dehydrated in a graded series of ethanol, followed by propylene oxide, and embedded in epoxy resin. Samples were cut at a thickness of 1 μm and stained with toluidine blue for light microscopic assessment. Samples for electron microscopy were cut at a thickness of 20 nm and stained with uranyl acetate and lead citrate.

Actual concentrations of bacteria for the animals inoculated with a high dose ranged from 7.5 × 10⁵ to 3.4 × 10⁷ CFUs for the wild-type strain, 2.3 × 10⁶ to 5.8 × 10⁷ CFUs for Ply⁻, 7.5 × 10⁵ to 8.1 × 10⁷ CFUs for PspA⁻ mutants, and 5.5 × 10⁵ to 5.5 × 10⁶ CFUs for PsaA⁻ mutants. The condition of the animals is listed in Table 1. Of the 15 animals inoculated with the high-dose wild-type strain, 3 died, as did 1 of the animals inoculated with the high-dose PspA⁻ mutants. Three of the animals inoculated with the high-dose wild-type strain, and 1 of those inoculated with the PspA⁻ mutant, became extremely ill and had to be killed 1 day after inoculation. Actual concentrations of bacteria for the low-dose S pneumoniae were 4.8 × 10³ to 7.5 × 10³ CFUs for the wild-type strain and 2.8 × 10³ CFUs for Ply⁻, 0.6 × 10³ CFUs for PspA⁻, and 7.5 × 10³ for PsaA⁻ strains. One of the chinchillas inoculated with low-dose PsaA⁻ died before the end of the experiment.

All animals, those inoculated with both high-dose and low-dose bacterial strains, had middle-ear effusions that were generally thin. Thick purulent effusions were seen overlying the round window membranes in all 12 animals inoculated with the high-dose wild-type strain, in 4 of 9 inoculated with the Ply⁻ mutants, 5 of 9 inoculated with the PspA⁻ mutants, and in none of the 7 inoculated with the PsaA⁻ mutants. Thick purulent effusions were seen overlying the round window membranes in 4 of the 5 animal inoculated with the low-dose wild-type bacterial strain, 4 of 6 inoculated with the Ply⁻ mutants, 1 of 6 inoculated with the PspA⁻ mutants, and in none of the 3 inoculated with the PsaA⁻ mutants.

A summary of inner ear pathologic findings are listed in Table 2. Bacterial infiltration of the round window membranes (Figure 1) was seen in 6 of 12 animals inoculated with the high-dose wild-type strains, 7 of 9 animals inoculated with Ply⁻ mutants, 0 of 9 inoculated with PspA⁻ mutants, and 0 of 7 inoculated with PsaA⁻ mutants. Inflammatory cell infiltration of the round window membrane (Figure 1) was seen in 5 of 12 animals inoculated with the wild-type strain, 7 of 9 inoculated with Ply⁻ mutants, 2 of 9 inoculated with PspA⁻ mutants, and 1 of 7 inoculated with PsaA⁻ mutants. Bacterial infiltration of the scala tympani (Figure 2) was seen in 5 of 12 inoculated with wild-type strains, 7 of 9 inoculated with Ply⁻ mutants, in none inoculated with 9 PspA⁻ mutants, and 0 of 7 inoculated with PsaA⁻ mutants. Inflammatory cell infiltration of the scala tympani (Figure 2) was seen in 5 of 12 animals inoculated with the wild-type strain, 7 of 9 inoculated with Ply⁻ mutants, 3 of 9 inoculated with PspA⁻ mutants, and 1 of 7 inoculated with PsaA⁻ mutants. Strial edema (Figure 2) and/or strial atrophy occurred in 5 of 12 animals inoculated with the wild-type strain, 5 of 9 inoculated with Ply⁻ mutants, 2 of 9 inoculated with PspA⁻ mutants, and 1 of 7 inoculated with PsaA⁻ mutants. Damage to (Figure 2 and Figure 3) and/or loss of inner and outer hair cells occurred in 3 of 12 animals inoculated with the wild-type strain, 5 of 9 inoculated with Ply⁻ mutants, 2 of 9 inoculated with PspA⁻ mutants, and 1 of 7 inoculated with PsaA⁻ mutants. Pathologic changes to the neurons, including bacterial infiltration (Figure 3), hemorrhage, inflammatory cell infiltration, or neuronal damage, was seen in 4 of 12 animals inoculated with the wild-type strain, 6 of 9 inoculated with Ply⁻ mutants, 2 of 9 inoculated with PspA⁻ mutants, and 1 of 7 inoculated with PsaA⁻ mutants.

In the animals inoculated with the low-dose bacterial strain, pathologic changes occurred only in those inoculated with the wild-type strain. Of the 5 animals inoculated with the wild-type strain, bacterial infiltration of the round window membrane and scala tympani was seen in 2, inflammatory cell infiltration of the round window membrane in 3, inflammatory cell infiltration of the scala tympani in 2, strial edema and/or atrophy in 1, hair cell damage and/or loss in 2, and neuronal damage in 1.

We have previously shown⁸ that bacteria and their toxins can pass from the middle ear into the inner ear in animals, resulting in inner ear damage and hearing loss. Hunter et al⁹ showed an association between high-frequency hearing loss and OM in children, even after middle-ear disease resolved and middle-ear dysfunction was excluded. A study⁵ of the consequences of chronic OM on inner ear function showed a correlation of the severity of sensorineural hearing loss with longer duration of middle-ear disease. A human temporal bone study of unilateral chronic OM found inner ear damage to be more severe in the ear with OM than in the contralateral ear.¹⁰

Widespread use of oral antibiotics has resulted in an alarming increase of antibiotic-resistant bacterial strains, increasing the potential for labyrinthitis, tympanogenic meningitis, and sensorineural hearing loss. Zapalac et al⁴ described 90 patients with suppurative complications of acute OM over a period of 7.5 years. They found that all but 2 cases of pneumococcal-related suppurative complications were attributable to resistant strains in the final 2.5 years, with resistant organisms isolated in 75% of patients.

Currently approved vaccines have proven to be only partially effective. The Pneumovax 23 valent polysaccharide vaccine does not protect children younger than 2 years,⁶ and conjugate vaccines have limited serotype protection.¹¹ This led investigators to consider other potential vaccine candidates, such as pneumococcal proteins.

Pneumococcal proteins facilitate important aspects of pneumococcal colonization and/or invasion and can therefore serve as targets for the development of novel therapies to treat pneumococcal diseases. There are several pneumococcal proteins that contribute to the virulence and pathogenicity of streptococcus pneumoniae, including Ply, a cytoplasmic cholesterol binding, pore-forming protein¹²; PspA, a membrane-bound protein that prevents compliment fixation^13,14; and PsaA, a surface protein that is a magnesium²⁺ and zinc²⁺ transporter, involved in growth and virulence.¹⁵

Although Ply is a potent cytotoxic agent, we found inner ear permeability and damage in animals inoculated with the Ply⁻ strains to be remarkably similar in virulence to the wild-type strain. Pneumolysin is located in the cytoplasm and is dependent on autolysin for its release. Sato et al¹⁶ showed that in the middle ear, pneumococcal OM pathogenesis is triggered principally by the inflammatory effects of intact and lytic cell wall products with, at most, a modest additional Ply effect.

Findings in the PspA and PsaA mutants were similar. Neither mutant seemed to pass through the round window membrane, and both mutants resulted in far less inner ear damage than either the wild-type strain or the Ply⁻ mutant. It must be acknowledged, however, that the animals were allowed to survive for only 2 days because those inoculated with the wild-type bacteria became sick and began to exhibit signs of labyrinthitis. It is possible that it may take the mutant bacteria longer to multiply to a level necessary to avoid clearance and enter the inner ear; however, the lack of virulence in these mutants may also be due to their missing proteins. The PspA protein is a cell surface protein that is protective against the host complement system.^14,15 It also has the ability to bind lactoferrin,¹⁷ an iron storage glycoprotein predominantly found in mucosal secretions, including those of the middle ear,¹⁸ and a study¹⁹ has shown that resistance to pneumococcal carriage is dependent on mucosal rather than systemic immunity. The PspA⁻ mutants have also been shown to have a decreased capacity to form biofilms.²⁰

Even though PspAs are antigenetically and structurally variable, they are immunologically cross-reactive,^21,22 and immunization with a single PspA stimulates broadly cross-reactive antibodies.²³ Because PspA is expressed by all clinically important capsular serotypes, it is particularly attractive as a vaccine candidate.²⁴

Although clinically significant mucosal responses are directed to PspA, the systemic immune system seems to mount the highest humoral and cellular immune responses to PsaA.²⁵ The PsaA protein is known to be a surface binding protein that inhibits complement activation.¹³ It has also been shown to play an important role in protection against pneumococcal carriage.²⁶ In nasopharyngeal epithelial cells, adherence of S pneumoniae is the primary step essential for its pathogenesis,²⁷ and Romero-Steiner et al²⁸ identified a PsaA putative binding domain contained within a 28 amino acid peptide (P4) that contributes to PsaA adherence to nasopharyngeal cells. Immunization with a combination of PspA and PsaA proteins have been shown to offer better protection against nasal carriage in mice than immunization with either protein alone.²⁷

Given the different roles of these proteins and the fact that they both seem to exhibit virulence factors that can have an impact on the inner ear, it may be interesting to explore a combination of these proteins in their role in the pathogenesis of OM and inner ear damage.

Correspondence: Patricia A. Schachern, BS, Room 226, Lions Research Building, 2001 Sixth St SE, Minneapolis, MN 55455 (schac002@tc.umn.edu).

Submitted for Publication: July 29, 2008; final revision received October 16, 2008; accepted November 13, 2008.

Author Contributions: All of the authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Schachern, Tsuprun, Briles, Paparella, and Juhn. Acquisition of data: Schachern, Tsuprun, Cureoglu, and Ferrieri. Analysis and interpretation of data: Schachern and Tsuprun. Drafting of the manuscript: Schachern, Tsuprun, and Briles. Critical revision of the manuscript for important intellectual content: Schachern, Tsuprun, Cureoglu, Ferrieri, Briles, Paparella, and Juhn. Administrative, technical, and material support: Schachern, Tsuprun, Cureoglu, Ferrieri, and Briles. Study supervision: Ferrieri, Paparella, and Juhn.

Financial Disclosure: None reported.

Funding/Support: This study was supported by National Institute on Deafness and Other Communications Disorders (grant R01 DC006452), the International Hearing Foundation, the Starkey Foundation, and the Hubbard Foundation.