Capsule Enhances Pneumococcal Colonization by Limiting Mucus-Mediated Clearance^{▿ †}

Mouse model of nasopharyngeal colonization.

Six-week-old, female C57BL/6J (wild type) or B6.129-S2-Igh-6^tm1Cgn/J (μMT) mice (Jackson Laboratories, Bar Harbor, ME) were housed in accordance with Institutional Animal Care and Use Committee protocols. μMT mice contain a targeted mutation in the heavy-chain locus of immunoglobulin M (IgM) and do not produce mature B cells or antibody (15). BALB/c mice were obtained from Taconic of Germantown, NY. Mice were colonized using a previously described model of nasopharyngeal colonization with S. pneumoniae (20). Briefly, groups of at least five mice per condition were inoculated intranasally without anesthesia with 10 μl containing 1 × 10⁷ to 3 × 10⁷ CFU of phosphate-buffered saline (PBS)-washed, mid-log-phase S. pneumoniae applied to each naris. At the time indicated, the animal was sacrificed, the trachea was cannulated, and 200 μl of PBS was instilled. Lavage fluid was collected from the nares for determination of viable counts of bacteria in serial dilutions plated on selective medium containing neomycin (5 μg/ml) to inhibit the growth of contaminants. The lower limit of detection for bacteria in lavage culture was 20 CFU/ml.

Neutrophil and complement depletion.

Monoclonal antibody (MAb) RB6-8C5, a rat anti-mouse IgG2b directed against Ly-6G on the surface of murine myeloid (and limited subpopulations of lymphoid) lineage cells, was purified from ascites of nude mice given the RB6-8C5 hybridoma (4, 11). To deplete neutrophils, 150 μg of MAb/animal was administered by intraperitoneal (i.p.) injection 24 h prior to intranasal challenge with bacteria. This dose was previously shown to result in peripheral blood neutropenia (<50 granulocytes/μl) for a period of at least 48 h (17). Controls were given the equivalent i.p. dose of total rat IgG (Sigma Chemical Co., St. Louis, MO).

Hypocomplementemia was induced by i.p. injection of 25 μg/animal of cobra venom factor (Quidel, San Diego, CA) in PBS 18 h prior to bacterial challenge. This procedure was previously shown to reduce levels of immunodetectable C3 to <3% of normal and result in a period of hypocomplementemia of at least 48 h (30).

Histology and immunofluorescence.

At the time indicated postinoculation, the animal was sacrificed and decapitated, and the head was fixed for 48 h in 4% paraformaldehyde in PBS. The head was then decalcified by serial incubations in 0.12 M EDTA (pH 7.0) over 1 month before freezing in Tissue-Tek O.C.T. embedding medium (Miles) in a Tissue-Tek Cryomold. Five-micrometer-thick sections were cut and stored at −80°C. Frozen imbedded tissue sections were stained with hematoxylin and eosin (H&E) following a 10-min fixation step in 10% neutral buffered formalin. Sections were then dehydrated in alcohol, cleared in xylene, and mounted in Cytoseal (Richard-Allan Scientific, Kalamazoo, MI).

For immunohistochemistry, sections were postfixed in 1:1 methanol-acetone at −20°C for 10 min followed by washing in distilled water (dH₂O). Endogenous peroxidase was blocked by incubation in 2.25% H₂O₂ in dH₂O for 15 min. Sections were blocked with avidin and biotin for 15 min each, followed by a further 10-min incubation in protein blocking reagent (PBR; Coulter/Immunotech, Miami, FL) to prevent nonspecific binding. Sections were then incubated in three steps in typing sera (Staten Serum Institute, Copenhagen, Denmark) at 4°C overnight, 1:500 in PBT (1× PBS, 0.1% bovine serum albumin, 0.2% Triton X-100), biotinylated goat anti-rabbit for 30 min, 1:200 in PBT, avidin-horseradish peroxidase-conjugated ABC reagent for 30 min (Vector Laboratories, Burlingame, CA), and the signal was developed using diaminobenzidine tetrahydrochloride (DAB) kit (Vector Laboratories). Sections were washed with PBS after each incubation step. After DAB staining, the slides were fixed in 10% NBF for 3 min followed by a 3-min wash in dH₂O. Sections were exposed to 3% acetic acid (pH 2.5) for 3 min, before being incubated with 1% alcian blue in 3% acetic acid (pH 2.5) for 30 min. After washing in water for 10 min, sections were counterstained with 0.1% nuclear fast red for 40 s and then dehydrated in alcohol, cleared in xylene, and mounted in Cytoseal. Bright-field imaging was performed on a Nikon E600 Eclipse microscope equipped with a liquid crystal (Micro-color RGB-MS-C, CRI Inc., Boston, MA) and a high-resolution charge-coupled device (CCD) digital camera (CoolSnap CF, Roper Scientific, Tucson, AZ) with Nomarski optics.

For immunofluorescent staining, tissue was postfixed with 1:1 acetone-methanol as described above, followed by blocking with PBR before addition of primary antibody, diluted 1:500 in PBT. Pneumococci were stained with typing sera as described above. Signal was detected with Cy3-conjugated species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA) incubated in a 1:400 dilution of PBT for 2 h at room temperature. After washing with PBS followed by dH₂O, sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes, Invitrogen, Carlsbad, CA) diluted 1:10,000 in dH₂O. All image analysis was carried out using IPLAB (Scanalytics, Fairfax, VA).

Human upper airway secretion binding assays.

Mucus samples from human upper airway secretions obtained as previously described were used in a modified solid-phase mucin binding assay (8, 25). Secretions were diluted 1:1 in sterile PBS, sonicated until homogenous, and applied to 96-well Polysorp plate (Nunc) by centrifugation at 250 × g for 3 min at room temperature (8). Following overnight incubation at 37°C, the unbound secretions were removed by washing with PBS and treated for an additional 3 h in sodium citrate buffer (50 mM, pH 6.0) with or without recombinant neuraminidase derived from Clostridium perfringens (25 U/ml; New England Biolabs, Beverly, MA). Bacterial cultures grown to mid-log phase (optical density at 620 nm [OD₆₂₀] of 0.4) were diluted in PBS to a density of 10⁵ CFU/ml and applied by centrifugation at 250 × g for 3 min at room temperature. Adherence was determined by incubation at 4°C for 60 min with gentle agitation. Supernatants and the first wash with PBS were removed for colony counts. Bacteria in the supernatant and first wash were considered to have not adhered to the well and were compared to the inoculum to calculate the proportion of bound or adherent bacteria. In the absence of fixed secretions (PBS-alone control), there was no significant adherence.

To generate a column to assess migration, mucins extracted from human upper airway secretions were covalently linked to acrylamide beads using UltraLink Biosupport according to the manufacturer's instructions (Pierce, Rockford, IL). Binding of mucins was monitored for saturation by assay of bound protein. Where specified, the mucin-treated beads were incubated with neuraminidase as described above. After equilibration with PBS, a 0.5-ml column of beads was loaded with 10⁷ CFU of washed, mid-log-phase bacteria and fractions of the flowthrough were collected for quantitative culture.

Adherence to primary murine cells.

Primary tracheal epithelial cells were isolated from C57BL/6 mice as described with two modifications (6). NuSerum (BD Biosciences) was used in differentiation medium in place of Ultroser G, and ITS+ tissue culture supplement (BD Biosciences) was used in the culture medium in place of insulin. Cells were cultured at an air-liquid interface for 3 weeks, and abundant ciliary activity was noted prior to adherence experiments. To assess the relative adherence of encapsulated and unencapsulated pneumococci, TIGR4 and TIGR4 cps were grown to OD₆₂₀ of 0.5 in tryptic soy broth, pelleted, and washed twice with sterile PBS. Bacteria (1 × 10⁸ CFU in 200 μl differentiation medium) were added to the apical chamber of the transwells and applied to the epithelial cells by centrifugation (150 x g for 5 min) prior to incubation at 37°C in 5% CO₂ for 2 h. Following incubation, monolayers were washed four times with sterile PBS. Cells were lifted by treatment with a 0.25% trypsin-1 mM EDTA solution (Gibco), and serial dilutions were plated for colony counting.

Capsule does not impact on opsonophagocytic clearance during colonization.

Histological examination of colonized nasal tissues confirmed that colonizing pneumococci induce a neutrophil influx into lateral nasal spaces by 1 day with a maximal response by 3 days postinoculation (see Fig. S1 in the supplemental material) (21, 35). Immunofluorescent staining of frozen tissue to detect bacteria demonstrated that these dense clusters of neutrophils have engulfed pneumococci but that not all pneumococci become associated with neutrophils (data not shown). To test whether neutrophil-mediated clearance accounted for the lower density of colonization by unencapsulated pneumococci, mice were treated with RB6-8C5, a rat MAB to murine Ly6.G, prior to intranasal challenge (11). This treatment effectively depleted neutrophils from peripheral blood and in tissue sections of colonized mice (17). If opsonophagocytic clearance were important during colonization, this treatment would be expected to enhance colonization by more readily opsonized unencapsulated pneumococci. However, there was no significant effect of RB6-8C5 treatment compared to that of controls on the density of TIGR4 cps at 2 days postinoculation (Fig. ​(Fig.3).3). Similarly, no effect on the colonization density of TIGR4 cps was observed following complement depletion by systemic administration of cobra venom factor prior to nasal challenge. In addition, genetically modified congenic mice (μMT) that fail to generate specific antibody did not demonstrate significantly enhanced colonization by TIGR4 cps compared to parental mice. These findings indicated that neutrophil-, complement-, or antibody-mediated clearance mechanisms do not contribute substantially to the lower density of colonization by unencapsulated mutants and left in question the role of capsule during colonization.

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FIG. 3.

Effect of neutrophils, complement, and functional antibody on colonization by an unencapsulated mutant. C57BL/6 mice were pretreated with RB6-8C5 to deplete neutrophils (or rat IgG control) or cobra venom factor (CoVF) to deplete complement (or vehicle control) prior to intranasal challenge with 10⁷ CFU of TIGR4 cps, and the density of colonization was assessed in quantitative culture of upper respiratory tract lavage fluid on postinoculation day 2. Colonization of congenic μMT mice was determined in parallel experiments. Values represent the mean of 5 to 10 mice/strain at each time point ± standard deviation. P > 0.05 (Kruskal-Wallis test with Dunn's post-test for multiple comparisons).

Effect of capsule on the dynamics of colonization.

To define the role of capsule in pneumococcal colonization, the events during the initial 2-day period postinoculation, during which the majority of the deficit in colonization by unencapsulated mutants occurs, were visualized in tissue sections. For TIGR4, initially (30 min) bacteria were confined to the lumen of nasal spaces, where they associate with amorphous, acellular material (Fig. ​(Fig.4A).4A). This lumenal material is mucus based on its staining with alcian blue, which identifies acidic mucopolysaccharides (Fig. ​(Fig.4C).4C). By 1 day postinoculation, these encapsulated pneumococci had transited to the mucosal surfaces, where they were found along the glycocalyx, a thin mucus-containing layer overlying epithelial cells (Fig. 4B and D). At later time points up to 14 days, pneumococci remained in the glycocalyx, indicating that this was the site of stable colonization (see Fig. S1 in the supplemental material).

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FIG. 4.

Early events in colonization showing transition from mucus to the epithelial surface for encapsulated pneumococcal strain TIGR4. (A and B) Frozen nasal tissue from C57BL/6 mice colonized for 30 min (A) or 2 days (B) and stained with type-specific sera detected with Cy3 secondary antibody (red) and DAPI (blue) superimposed with Normarski bright-field optics to show the epithelial border. (C and D) Frozen nasal tissue from C57BL/6 mice colonized for 30 min (C) or 2 days (D) stained with type-specific sera and detected with horseradish peroxidase-conjugated secondary antibody and DAB substrate (brown), alcian blue (pH 2.5) (blue), and nuclear fast red (red). Arrows indicate bacteria. Scale bar, 10 μm.

TIGR4 cps was also seen initially in the lumenal mucus (30 min), but unlike the encapsulated parent was heavily agglutinated in mucus (Fig. ​(Fig.5A5A versus B). Similar results were noted when isolates P303 and P303 cps were compared at 30 min postinoculation (Fig. ​(Fig.5C5C versus D). When rare unencapsulated mutants of TIGR4 were seen later at 20 h postinoculation, these were still confined to lumenal mucus rather than along the epithelial surface, suggesting an inability to transit to the epithelial surface as occurred for the encapsulated parent (Fig. ​(Fig.5E5E versus F).

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FIG. 5.

Unencapsulated pneumococci remain trapped within lumenal mucus. (A and B) H&E-stained frozen nasal tissue from C57BL/6 mice 30 min postinoculation with (A) TIGR4 or (B) TIGR4 cps. Only the unencapsulated mutant is heavily agglutinated in mucoid material in the lumen. Pneumococci are marked by arrows. (C to F) Frozen nasal tissue from C57BL/6 mice comparing P303 (C) and P303 cps (D) or TIGR4 (E) and TIGR4 cps (F) at 30 min (C and D) or 20 h postinoculation (E and F) using staining with type-specific sera detected with Cy3 secondary antibody (red) and DAPI (blue). Tissue autofluorescence (green) reveals the epithelial border. Scale bar, 10 μm.

We considered whether these in vivo observations could be explained by increased adherence of encapsulated pneumococci to epithelial cells. However, when the abilities of TIGR4 and TIGR4 cps to adhere to ciliated monolayers of primary mouse tracheal epithelial cells in culture were compared, the presence of capsule was associated with a >500-fold decrease in the percentage of the inoculum bound (data not shown).

This work was supported by grants from the U.S. Public Health Service to J.N.W. (AI44231 and AI38446) and to the Bacterial Respiratory Pathogen Research Unit (NO1 AI30040) and The Morphology Core of the Center for the Molecular Studies of Liver and Digestive Disease (P30 DK50306) and from the Howard Hughes Medical Institute (K.C.).