Pneumococcal Neuraminidase A (NanA) Promotes Biofilm Formation and Synergizes with Influenza A Virus in Nasal Colonization and Middle Ear Infection

ABSTRACT

Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae, in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens.

INTRODUCTION

Streptococcus pneumoniae (the pneumococcus) is a widespread colonizer of the human nasopharynx and is found in as many as 85% of children under the age of 4 years (1). While this colonization is typically asymptomatic and self-limiting, it is also the requisite precursor for most if not all pneumococcal diseases (2, 3). Otitis media (OM) specifically is frequently caused by S. pneumoniae, is one of the most significant public health burdens, with as many as 700 million cases occurring worldwide each year, and disproportionately affects preschool-age children (4, 5). Coinfection with respiratory tract viruses, particularly influenza A virus (IAV), is epidemiologically and experimentally linked to the transition of S. pneumoniae from a colonizer that does not cause symptoms to an otopathogen (6–13). The role of specific virulence factors in this viral-bacterial interaction, however, remains to be fully explored.

Neuraminidases are widely expressed as virulence factors by a number of bacteria and viruses, including both S. pneumoniae and IAV (14, 15). These proteins possess sialidase activity to catalyze the cleavage of terminal sialic acid residues from glycoconjugates. The influenza virus neuraminidase has a well-established role in the pathogenesis of IAV infection, and inhibitors directed at this target are currently first-line therapeutics in the United States (16). In addition, IAV neuraminidase activity has been shown to interact synergistically with S. pneumoniae. Indeed, pneumococcal adherence in vitro and pulmonary infection in vivo are increased during coinfection with IAV (17–19). This effect is reliant, at least in part, on the activity of the viral neuraminidase, as IAV neuraminidase inhibitors have been shown to protect against pneumococcal pneumonia in mice independently of their effect on viral replication (17).

S. pneumoniae expresses as many as three neuraminidases, of which NanA is the most highly conserved, expressed, and active at physiologic pH (20–24). The modification of host glycoconjugates by NanA plays a multifactorial role in pneumococcal pathogenesis. It is most well-known that the sialidase activity of NanA exposes desialylated residues on host glycans and mucins to which S. pneumoniae can adhere (25, 26). In addition, NanA desialylates host immune factors, thereby potentially promoting persistence (22, 27). Further, the sialic acid released by neuraminidase activity can act as a carbon source as well as a diffusible signal for enhanced pneumococcal proliferation both in vivo and in vitro (28, 29). Interestingly, recent evidence has also indicated that neuraminidase activity may impact biofilm formation. Indeed, reductions in biofilm biomass in vitro have been observed in neuraminidase-deficient and neuraminidase inhibitor-treated S. pneumoniae as well as in Pseudomonas aeruginosa and Tannerella forsythia (30–33). These findings are generally supported by animal models, where NanA-deficient pneumococci are impaired in their ability to cause nasal colonization, otitis media, pneumonia, and bacteremia (24, 34). There are, however, conflicting findings (22, 35), suggesting a complex role for NanA in pneumococcal pathogenesis that is still being appreciated.

On the basis of our findings, it is evident that IAV neuraminidase activity significantly contributes to pneumococcal colonization and disease. Left unclear, however, is the role of the pneumococcal neuraminidase NanA in this synergistic interaction, particularly as it relates to nasal colonization and middle ear infection. To assess this, we utilized a mutant strain of S. pneumoniae that does not express NanA in our previously established mouse model of S. pneumoniae and IAV coinfection (12). Our studies identified that prior IAV infection potentiates but cannot completely restore the ability of NanA-deficient pneumococci to cause nasal colonization and middle ear infection, suggesting that the activity of the viral neuraminidase was insufficient to fully complement the absent expression of NanA. Additionally, we demonstrated in vitro that pneumococcal adherence but, interestingly, not biofilm viability can be reduced by a neuraminidase inhibitor. Taken together, these results imply a potential intrinsic role for NanA distinct from its enzymatic activity in pneumococcal pathogenesis.

RESULTS

Nasal colonization is impaired in NanA-deficient pneumococci and is partially restored by influenza virus coinfection.

To assess the role of the pneumococcal neuraminidase NanA in nasal colonization during coinfection with influenza virus, mice were infected intranasally with IAV and then 4 days later with S. pneumoniae EF3030 or an isogenic nanA-deficient mutant (S. pneumoniae nanA). S. pneumoniae nanA was confirmed to grow similarly to S. pneumoniae EF3030 in planktonic culture, to possess minimal neuraminidase enzymatic activity, and to not express NanA when NanA expression was assessed by Western blotting (data not shown). As predicted from prior studies (24, 34), the magnitude of nasal colonization by S. pneumoniae nanA was significantly reduced at both day 2 and day 4 after bacterial infection relative to that by S. pneumoniae EF3030 (P < 0.0001). In recent work, we showed that IAV titers were not significantly affected during this type of coinfection with S. pneumoniae EF3030 (12). Coinfection with IAV significantly increased the nasal colonization density of both S. pneumoniae EF3030 and S. pneumoniae nanA (P < 0.0001) (Fig. 1A). This, however, was not sufficient to enable S. pneumoniae nanA to colonize the nasopharynx to the same magnitude as parent strain S. pneumoniae EF3030 during coinfection with IAV. Indeed, during viral coinfection, the nasal pneumococcal burden of S. pneumoniae nanA was approximately 5-fold lower than that of S. pneumoniae EF3030 at both time points studied (Fig. 1A). Taken together, these findings suggest that expression of both the viral and pneumococcal neuraminidases is required for complete synergistic activity between the two pathogens in the nasopharynx.

FIG 1.

NanA is involved in nasal colonization and middle ear infection by S. pneumoniae in the presence and absence of influenza A virus coinfection. Mice were infected intranasally with IAV A/Puerto Rico/8/34 (H1N1)-GFP, followed 4 days later by intranasal inoculation with either S. pneumoniae EF3030 or S. pneumoniae EF3030 nanA. Data represent the total pneumococcal burden in tissue homogenates from the nasopharynx (A) and middle ear (B) from days 2 and 4 after bacterial infection, corresponding to days 6 and 8 after viral infection, respectively. Each data point denotes the result for a single nasopharynx or middle ear. The solid horizontal bars indicate the geometric mean, and the dotted lines denote the limit of detection of the assay. Data were pooled from two replicate experiments. Statistical analysis was performed using a Kruskal-Wallis comparison followed by a post hoc Mann-Whitney U test (**, P < 0.01; ***, P < 0.001).

Influenza A virus potentiates middle ear infection by NanA-deficient pneumococci to a level below that by the parental strain.

IAV infection has previously been shown to increase the magnitude of middle ear infection by S. pneumoniae EF3030 (10, 12). To determine whether pneumococcal NanA was required in this interaction or if its absence could be fully complemented by coinfection with IAV, the middle ear bacterial burden in singly and coinfected mice was enumerated. The magnitude of middle ear infection by S. pneumoniae nanA was significantly reduced relative to that by the wild type at day 4 (P < 0.0001) (Fig. 1B), illustrating an important role for NanA in this process even in the absence of influenza virus. Coinfection with IAV significantly increased the magnitude of middle ear infection by both wild-type and NanA-deficient pneumococci at days 2 and 4 after bacterial infection (P = 0.0031 and P < 0.0001, respectively, for the wild type and P = 0.0012 and P < 0.0001, respectively, for S. pneumoniae nanA) (Fig. 1B). Despite this, the magnitude of middle ear infection by the S. pneumoniae nanA mutant was still significantly less than that by the parental strain following coinfection with IAV (P < 0.0001). Taken together, these data indicate that NanA has an intrinsic role in pneumococcal middle ear infection that cannot be fully complemented by coinfection with neuraminidase-expressing IAV.

NanA enzymatic activity is important for pneumococcal adherence to epithelial cells.

As IAV coinfection did not fully restore colonization and middle ear infection by NanA-deficient pneumococci in vivo, we postulated that NanA may contribute to colonization independently of its enzymatic activity. This would account for the inability of IAV coinfection to fully complement the absence of NanA expression. Therefore, we tested the role of NanA enzymatic activity in bacterial adherence. NanA expression is upregulated following exposure to epithelial cells, and adherence to these cells is a critical and requisite step in pneumococcal colonization (36–38). We tested the role of NanA sialidase activity in this process using N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA), a competitive transition-state analog of sialic acid that acts as a potent neuraminidase inhibitor. Utilizing the fluorogenic sialic acid analog 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUAN) as an indicator of neuraminidase activity, we determined that maximal inhibition occurred at approximately 300 μg/ml DANA (Fig. 2A), a finding comparable to prior findings (31). Of note, DANA also inhibited the residual neuraminidase activity of S. pneumoniae nanA, likely attributable to the inhibition of NanB as well (39). Adherence to cells of the Detroit 562 cell line, an immortalized human nasopharyngeal epithelial carcinoma cell line, was significantly impaired for S. pneumoniae nanA compared with that for the parental strain. A similar magnitude of impairment was observed for S. pneumoniae EF3030 treated with 300 μg/ml DANA (Fig. 2B). These data indicate that NanA, specifically, its enzymatic activity, is a determinant of pneumococcal adherence.

FIG 2.

The sialidase activity of NanA is inhibited by DANA and contributes to epithelial adherence in vitro. (A) Inhibition of NanA sialidase activity in lysates of S. pneumoniae EF3030 and S. pneumoniae EF3030 nanA by 15 min of incubation with DANA at various concentrations. The assay was performed using the fluorogenic sialic acid analog 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. Each data point represents the mean ± SEM. Each sample was measured in triplicate, and the experiment was performed twice. (B) Adherence of S. pneumoniae EF3030 and S. pneumoniae EF3030 nanA incubated with DANA (300 μg/ml) or the vehicle control after 60 min, expressed as the percent adherence relative to the inoculum size. Each experiment contained at least three biological replicates and was repeated six times. Statistical analysis was performed by a one-way analysis of variance with a Bonferroni post hoc test of significance (*, P < 0.05; ***, P < 0.0001).

NanA is involved in pneumococcal biofilm formation independently of its enzymatic activity.

To continue investigating how NanA may specifically impact pneumococcal pathogenesis, we next assessed its effect on biofilm formation in vitro. Biofilms are surface-attached microbial communities encased in an extracellular matrix that possess well-described roles in both nasal colonization and middle ear infection (40, 41). NanA has previously been shown to be involved in pneumococcal biofilm formation in association with human epithelial cells (31). We hypothesized that NanA may also be involved in pneumococcal biofilms independently of its sialidase function on host epithelial cells, as has been observed in biofilms of Pseudomonas aeruginosa (32).

Utilizing a previously established model of in vitro biofilm viability on an abiotic polystyrene surface (12, 42), we found that the viability of biofilms formed by NanA-deficient pneumococci was significantly reduced by 3.25-fold relative to that of biofilms formed by the parental strain (P < 0.0001) (Fig. 3A). While epithelial cells and their sialylated glycoconjugates were not present in this model, the growth medium itself (Todd-Hewitt broth supplemented with 0.5% yeast extract, 10% heat-inactivated horse serum, and 2,500 U/ml catalase [THY⁺ medium]) was found by a thiobarbituric acid assay to contain numerous sialylated residues (Fig. 3B). To determine whether this biofilm defect persisted in the absence of both epithelial cells and sialylated moieties, we performed a similar experiment in growth medium (YSK) confirmed to be sialic acid free (Fig. 3B) (29). Interestingly, even in the absence of detectable sialic acid, NanA-deficient pneumococci similarly exhibited a significant 3.25-fold reduction in biofilm viability (P = 0.0003) (Fig. 3C). Further, a biofilm defect could not be induced in wild-type pneumococci grown in sialic acid-replete medium and treated with a neuraminidase inhibitor at concentrations sufficient to inhibit enzymatic activity (Fig. 3D), mirroring the findings of a recent study of S. pneumoniae clinical isolates (43).

FIG 3.

NanA contributes to in vitro biofilm viability independently of its sialidase activity. (A) Viability of S. pneumoniae EF3030 and S. pneumoniae EF3030 nanA from 4-h static biofilms grown in THY⁺ medium. (B) Sialic acid content of THY⁺ medium and YSK medium measured by a modified thiobarbituric acid assay, as described in Materials and Methods. Bars represent the means ± SEMs. Each measurement was made in triplicate, and the experiment was repeated three times. (C) Viability of S. pneumoniae EF3030 and S. pneumoniae EF3030 nanA from 4-h static biofilms grown in sialic acid-free YSK medium. (D) Static biofilm viability of S. pneumoniae EF3030 and S. pneumoniae EF3030 nanA in the presence of increasing concentrations of DANA. Biofilm viability was normalized to that for the untreated control and expressed as a percentage of that for the untreated control. Statistical analysis was performed using a one-way analysis of variance with a post hoc Dunnett's test (*, P < 0.05). Each experiment contained at least three biological replicates and was repeated at least twice. (E) Viability of S. pneumoniae opaque and transparent phase variants (confirmed to contain >90% colonies in the same phase) from 4-h static biofilms grown in sialic acid-replete THY⁺ medium. For all biofilm assays, bars denote the mean ± SEM of pooled data from the experiments. Unless indicated otherwise, all statistical analyses were performed using a two-tailed Student's t test (**, P < 0.01; ***, P < 0.001).

To further confirm these findings, we also examined the viability of transparent and opaque phase variants of S. pneumoniae within biofilms; these subpopulations of phase variants are typically associated with nasal colonization and disease, respectively (44). Specifically, isolates in the transparent phase have previously been shown to exhibit increased biofilm viability (12). Independently, NanA expression and activity have also been shown to be significantly increased in the transparent phase (22). To determine if the enzymatic activity of NanA contributed to the observed difference in biofilm viability between the two phases, biofilms of phase variants of S. pneumoniae EF3030 confirmed to contain >90% of colonies in the same phase were assessed in sialic acid-free medium (YSK). In this model, the biofilm viability of the transparent variant remained significantly increased relative to that of the opaque variant even in the absence of sialic acid (Fig. 3E). Taken together, these findings suggest that the role of NanA in pneumococcal biofilm formation by EF3030 is independent of its enzymatic sialidase activity. Further work is needed to determine the specific nonenzymatic contributions of NanA to this process and whether this finding contributes to the incomplete ability of IAV coinfection to fully restore nasal colonization and middle ear infection by NanA-deficient pneumococci to the levels observed for the parental strain.

DISCUSSION

Coinfection with S. pneumoniae and IAV is linked both experimentally and epidemiologically with the development of OM (6, 7, 45). The activity of the viral neuraminidase altering the respiratory and eustachian tube epithelium has long been proposed to be a mechanism by which preceding IAV infection potentiates the initiation of this disease by pneumococci (15, 46). The contributions of the bacterial neuraminidase to this process, however, are less well appreciated. In this study, we examined the role of and potential mechanisms whereby the primary pneumococcal neuraminidase, NanA, interacts with IAV during the exacerbation of nasal colonization and middle ear infection.

Extensive prior data illustrate that S. pneumoniae utilizes the sialidase activity of NanA to strip away sialic acid moieties, thereby enabling it to adhere to host cells (25, 26). Reflecting this, we observed in this study that an S. pneumoniae strain deficient in the expression of NanA was impaired in its ability to cause nasal colonization and middle ear infection in vivo, as has been similarly observed in outbred mice and chinchillas (24, 34), respectively. Of note, however, these findings are not universal in all animal models (22, 47, 48) and may reflect differences in pneumococcal serotypes or model species. Indeed, the sialic acid content can vary extensively between animal models (49). More generally, this discordance of the findings in the literature is also reflective of our adjusting appreciation for the numerous roles of NanA in pneumococcal pathogenesis. As such, the significant reduction in the magnitude of middle ear infection caused by the S. pneumoniae nanA mutant even during infection with a single pathogen is likely indicative of the roles of this virulence factor both in modifying the receptor complement of the eustachian tube and, likely, in altering host immune factors (22, 50). Illustrating this clinically, neuraminidase activity is frequently detected in the middle ear effusions of children with pneumococcal OM (51).

In addition to its canonical role in enhancing pneumococcal adherence during coinfection, the contributions of the IAV neuraminidase have recently been expanded to include the released sialic acid acting as a signaling molecule to induce pneumococcal outgrowth both in vitro and in vivo (18, 29). In the study presented here, we further contribute findings indicating that the pneumococcal neuraminidase NanA itself is not a redundant bystander in this process but, rather, is required for the full synergistic interaction between these two pathogens. Indeed, we show that while coinfection with IAV increases the levels of both nasal colonization and middle ear infection by NanA-deficient pneumococci, it is not to wild-type levels. Interestingly, this contrasts somewhat with the findings of King et al., who observed that NanA was not involved in the outgrowth of pneumococci in the lungs during IAV coinfection (48). This difference is likely tissue niche specific, particularly as NanA has been shown to be expressed at levels between 15- and 22-fold higher in the nasopharynx than in the lungs (36).

The relative inability of IAV coinfection to fully restore the nasal colonization and middle ear infection levels of the ΔnanA mutant to wild-type levels may stem from one or both of two potentialities. First, this may result from an incomplete ability of IAV neuraminidase activity to fully complement the absent enzymatic activity of NanA. Reflecting this possibility, the desialylation of airway epithelial cells appears to be maximal during coinfection (18, 50). Conversely, however, IAV preinfection of A549 epithelial cells was previously shown to be sufficient to restore wild-type adherence in S. pneumoniae nanA (15). A similar result was achieved by pretreatment of epithelial cells with exogenous neuraminidase (47). We are investigating this possibility. Second, as an alternative hypothesis, NanA may be involved in pneumococcal pathogenesis in a manner that would not be supplemented by the activity of the IAV neuraminidase. Supporting this hypothesis, Uchiyama et al. reported a critical, nonenzymatic role for NanA in brain endothelial cell invasion. The researchers identified, in addition to the C-terminal catalytic domain, an N-terminal lectin-like adhesion domain that was required for the full virulence of the bacteria (52). Interestingly, another pneumococcal exoglycosidase, the β-galactosidase BgaA, has also been shown to participate in cellular adherence independently of its enzymatic activity (53).

Adherence to epithelial cells represents a critical first step in pneumococcal pathogenesis (37). The sialidase activity of NanA has been shown to extensively modify the carbohydrate moieties of chinchilla tracheal and eustachian tube epithelial cells, and most, though not all, studies have reported reduced adherence by NanA-deficient pneumococci (22, 26, 30, 46, 47, 50). Utilizing a neuraminidase inhibitor, we demonstrated that the sialidase activity of NanA and not simply its expression was required for wild-type adherence. As the IAV neuraminidase exhibits an enzymatic function similar to that of the pneumococcal neuraminidase (54), these findings correlate well with previously published data in which an IAV neuraminidase inhibitor was observed to reduce pneumococcal adherence to IAV-infected airway epithelial cells (17).

While we confirmed that the role of NanA in the adherence of S. pneumoniae EF3030 was dependent on its enzymatic activity, whether a similar role exists during biofilm formation, a downstream step in pneumococcal colonization and disease, remained unclear. Parker et al. have reported the inhibition of pneumococcal biofilm formation in vitro during coculture with epithelial cells following treatment with several neuraminidase inhibitors (31). As the sialic acid released from cellular glycoconjugates can also independently stimulate biofilm growth (29), the specific role of NanA in this process is still difficult to identify. Neuraminidases of both P. aeruginosa and Porphyromonas gingivalis have been implicated in biofilm formation independently of epithelial cells (32, 55). In the present study, we similarly identified a role for the NanA neuraminidase in pneumococcal biofilms. Interestingly, however, this could not be inhibited by treatment with a neuraminidase inhibitor or by growth in sialic acid-free medium, indicating a nonenzymatic role for NanA in this process. Lending support to this hypothesis, Brittan et al. recently noted that biofilm formation by Lactococcus lactis was enhanced by in situ complementation with pneumococcal NanA and that this effect was not reversible by treatment with neuraminidase inhibitors (30).

Based on these findings, we propose a revised model of S. pneumoniae-influenza A virus coinfection wherein the pneumococcal neuraminidase NanA potentially both is complemented by the viral neuraminidase and also independently contributes to nasal colonization and middle ear infection. Specifically, the data indicate that NanA sialidase activity contributes to pneumococcal adherence in vitro, a feature which could potentially be supplemented by coinfection with neuraminidase-expressing viruses, as has been suggested previously (17). Conversely, maximal pneumococcal biofilm viability in vitro was dependent on NanA but independent of its enzymatic activity. This suggests a distinct role for NanA in pneumococcal pathogenesis that may not necessarily be fully complemented by IAV coinfection, as was observed in our in vivo nasal colonization and middle ear infection data. In sum, these findings establish an important role for the pneumococcal NanA during coinfection with IAV and further illustrate the complex interactions occurring between even analogous virulence factors of these two pathogens in the context of nasal colonization and middle ear infection.

MATERIALS AND METHODS

Infectious agents, growth conditions, and materials.

S. pneumoniae EF3030, a serotype 19F nasopharyngeal isolate, was used in all studies. This strain has been shown to colonize the mouse nasopharynx without causing lethal systemic disease (56, 57). Phase variants of the S. pneumoniae EF3030 strain containing >90% of colonies in the same phase were isolated as described previously (12). The S. pneumoniae EF3030 nanA-deficient strain used in this study (S. pneumoniae nanA) was generously provided by David Briles and has been described elsewhere (58). The IAV strain used in this study, influenza virus A/Puerto Rico/8/34-GFP (H1N1), was provided by Adolfo García-Sastre (59). This is a genetically modified strain which expresses the green fluorescent protein (GFP) gene (gfp) and causes mild disease in mice. Bacterial and viral strains were propagated and maintained as described previously (12).

Coinfection model.

All animal experiments were approved by the Wake Forest University Health Science Institutional Animal Care and Use Committee. Coinfection experiments were performed as described previously (12). Briefly, 6- to 8-week-old female BALB/c mice (The Jackson Laboratory) were inoculated intranasally with 3 × 10³ 50% tissue culture infective doses of IAV or the vehicle control, followed 4 days later by intranasal inoculation of 5 × 10⁶ CFU of S. pneumoniae or the vehicle control. Nasal and middle ear bacterial burdens were enumerated by tissue homogenization, serial dilution, and the plate count method at days 2 and 4 after bacterial infection.

Neuraminidase activity.

Neuraminidase activity was assessed using the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUAN), essentially as described previously (22, 24). MUAN (0.35%, wt/vol) was resuspended in 0.25 M sodium acetate buffer (pH 7), aliquoted, and stored at −20°C. For the assay, S. pneumoniae was grown in brain heart infusion broth (Becton, Dickinson) supplemented with 10% heat-inactivated horse serum and 10% catalase (2,500 U/ml; Worthington) until mid-logarithmic phase. S. pneumoniae was then centrifuged at 3,600 × g at 4°C, resuspended in Tris-HCl (pH 8), and freeze-thawed three times in liquid nitrogen, and the lysates were stored at −80°C. To measure neuraminidase activity, 10 μl of lysate was mixed with 10 μl of MUAN, and the mixture was incubated for 2 h at 37°C. The reaction was stopped by the addition of 50 mM sodium carbonate (pH 9.6). Fluorescence was detected using a POLARstar Omega plate reader (BMG Labtech) with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The background mean fluorescence intensity of the Tris-HCl vehicle incubated with MUAN was subtracted from each sample reading. Each reaction was measured in triplicate, and the experiment was repeated twice. Where indicated, the competitive neuraminidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA), a sialic acid transition state analog, was added to the bacterial lysates at various concentrations, and the mixture was incubated at 37°C for 15 min prior to incubation with MUAN.

Adherence assay.

The adherence of S. pneumoniae EF3030 and S. pneumoniae nanA to cells of the Detroit 562 cell line (ATCC CCL-138), an immortalized human nasopharyngeal epithelial carcinoma cell line, was assessed after 60 min of adherence, as described previously (12). Where indicated, the vehicle control or the neuraminidase inhibitor DANA (300 μg/ml) was added prior to incubation of the bacteria with epithelial cells. Each experiment contained at least three biological replicates and was repeated six times.

Biofilm assay.

The biofilm assay was performed in Todd-Hewitt broth supplemented with 0.5% yeast extract, 10% heat-inactivated horse serum, and 2,500 U/ml catalase (THY⁺ medium) essentially as described previously (12). Where indicated, pneumococci were mixed with 300 μg/ml DANA prior to inoculation into the biofilm wells. Biofilm assays were also performed using sialic acid-free medium (YSK) (29). Each experiment contained at least three biological replicates and was repeated a minimum of two times.

Sialic acid assay.

The sialic acid content was determined by a modified thiobarbituric acid assay (60–62). In this assay, 200 μl of sample was oxidized by incubation with 100 μl 0.2 M sodium periodate–9 M H₂SO₄ for 30 min at 37°C. Oxidation was stopped by addition of 1 ml sodium arsenite (10%, wt/vol) in 0.5 M sodium sulfate–0.05 M H₂SO₄. The chromophore was then developed by addition of 3 ml 2-thiobarbituric acid (0.6%, wt/vol) in 0.5 M sodium sulfate and boiling for 15 min. The solution was cooled and mixed 1:1 in acid butanol (5%, vol/vol), and the optical density at 549 nm (OD₅₄₉) and the OD₅₃₂ of the butanol fraction were determined. The sialic acid content (in micromoles), corrected for the presence of 2-deoxyribose, was calculated by the following equation: [(0.021 × OD₅₄₉) − (0.0078 × OD₅₃₂)] × total reaction volume (62). Each sample was measured in triplicate, and the experiment was repeated three times.

Statistical analysis.

Nasal colonization and middle ear bacterial infection were compared pairwise using a Mann-Whitney U test. The values for samples with bacterial counts below the limit of detection were plotted at the limit of detection for graphing and statistical analysis. The percent adherence of pneumococci to epithelial cells relative to the inoculum size was compared using a one-way analysis of variance with a Bonferroni posttest. Biofilm viability was analyzed using Student's t test, with the exception of DANA-treated biofilms, for which viability was normalized to that for untreated wells and the results were analyzed using a one-way analysis of variance with a post hoc Dunnett's test of significance. Where indicated, fold change refers to the geometric mean. Statistical significance was assessed according to conventional limits (P < 0.05). Statistical analyses were performed using GraphPad Prism (version 5.01) software (GraphPad Software).