INTRODUCTION

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Streptococcus pneumoniae is an important human pathogen that causes an array of diseases including otitis media, pneumonia, meningitis, and bacteremia. The pneumococcus is a component of the normal microflora in the nasopharynx, with colonization beginning shortly after birth (2). Colonization usually results in asymptomatic carriage within the nasopharynx, which subsequently serves as the main reservoir for pneumococci causing infections in children, the elderly, the immunocompromised, and individuals suffering from chronic disease (2). Because this nasopharyngeal reservoir of bacteria is so important to the dissemination and initiation of infection, colonization is an important target for the prevention of pneumococcal disease.

A number of S. pneumoniae components have been implicated in the colonization process. Among these are neuraminidase, SpxB (pyruvate oxidase), and the choline binding proteins CbpA (also referred to as PspC and SpsA [11, 25]), CbpD, CbpE, CbpG, LytB, and LytC. Mutants altered in the expression of each of these proteins show decreased colonization in animal models (24, 37, 43, 46). An increase in teichoic acid expression, along with a concomitant decrease in capsule expression, is correlated with an enhanced ability of transparent-phase variants to colonize (32, 50). Additional factors that may be involved in colonization have been suggested on the basis of in vitro adherence assays (AmiA, PlpA, PsaA, and cell wall components) and protection studies (PsaA and PspA) (5, 7, 8, 15, 16, 22, 55, 56).

In systemic infections, there is an absolute requirement for the polysaccharide capsule, which functions to inhibit complement-mediated opsonophagocytosis (3, 12, 27, 54). Virulence levels have been previously reported to correlate directly with the amount of capsule produced (32, 35), although these results have not been confirmed with isolates containing defined mutations. A role for the capsule in colonization has not been described, and in vitro studies have suggested that it may interfere with this process (1, 17, 32, 42, 44, 48). The ability of capsule-specific antibodies to reduce carriage, however, suggests that the capsule is expressed during nasopharyngeal colonization (18, 34, 36). Moreover, the varied ability of encapsulated strains of different serotypes to colonize the nasopharynx suggests some influence of the capsule on colonization (2, 14, 49, 50, 56). Here, we describe the requirement for capsule during nasopharyngeal colonization of mice and the effects of reduced capsule levels on both colonization and systemic infections.

	MATERIALS AND METHODS

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Bacteria and growth conditions. The strains and plasmids used in these studies are shown in Table 1. S. pneumoniae strains were grown in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (Difco) (THY) at 37°C or on blood agar base no. 2 (Difco) supplemented with 3% sheep erythrocytes (Colorado Serum Company, Denver, Colo.) at 37°C in 5% CO₂. Erythromycin was used at 0.3 µg/ml, and streptomycin was used at 100 µg/ml. For opacity determinations, strains were grown at 37°C in a candle jar on tryptic soy medium (Difco) plates containing 1% Bacto Agar (Difco) onto which 100 µl of catalase (5,000 U) was spread (50, 51).

DNA techniques. S. pneumoniae was transformed by induction with competence factor CSP-1 (28), as previously described (26). Plasmid DNA used for transformations was isolated using the alkaline lysis method (6). Chromosomal DNA was purified using Qiagen Genomic Tips (Qiagen, Inc., Valencia, Calif.). Insertion-duplication and deletion mutations were generated as previously described (26, 27, 58). The presence of the mutations was confirmed by Southern blot analyses for insertions and by PCR for deletions, as previously described (27). Using the digoxigenin labeling and detection system (Boehringer Mannheim, Indianapolis, Ind.), probes were generated by incorporation of digoxigenin-11-dUTP-labeled nucleotides during PCR amplification with Taq polymerase (Sigma). The deletion plasmid pCV182 was generated by cloning restriction fragments flanking the desired deletion into pJY4164 (Table 1). The cps3UMtnpA deletion mutant AM179 was obtained following transformation of pCV182 into S. pneumoniae without selection for the Em^r marker. Deletion mutants occurring as the result of allelic replacement were identified by PCR of pooled isolates (27) and confirmed by Southern blot analysis. The AM188 mutation was repaired by transformation with pJD380 without selection for integration of the plasmid (20). Repair of the mutation in isolates exhibiting the mucoid parental colony morphology was confirmed by sequence analysis. Sequencing was performed by the University of Alabama at Birmingham Automated Sequencing Facility. Primers used for sequence analysis were Cps3D-4 (5'-ATCGCGTGTATAGAGTTTTTCTTG-3'; bp 2170 to 2193), Cps3D-8 (5'-GCTTTGGTTACGGAGGGTATTGC-3'; bp 1781 to 1803), and Cps3D-11 (5'-GTATACATAAAAATTATTTCCCC-3'; bp 2212 to 2234). Base pair numbers correspond to the published cps3D sequence (19). The 7.5-kb deletion mutation in AM1000 was made by transformation of D39 with a PCR fragment containing the deletion generated from R36A chromosomal DNA using primers C2ups-3 (5'-GTCTATCTCTATCAACTTTTC-3'; bp 1019 to 1039) and Cps2I-1 (5'-CTGAATTTGTCCCAATAAC-3'; bp 11885 to 11897). Base pair numbers correspond to the published sequence of the type 2 capsule locus (31). All primers were obtained from Oligos Etc. (Wilsonville, Oreg.).

Capsule and teichoic acid measurements. Quellung (agglutination) reactions were performed using capsule-type-specific antisera (Statens Seruminstitut, Copenhagen, Denmark). For capsule measurements, cultures were grown to a density of 3 × 10⁸ CFU/ml in THY at 37°C. Cell-associated capsule production by type 3 strains was quantified using the Stains-All assay for detecting acidic polysaccharides (41) or by an indirect enzyme-linked immunosorbent assay (ELISA). For ELISAs, duplicate samples of heat-killed cells (65°C, 30 min) were serially diluted on a microtiter plate and incubated overnight at 4°C. Capsule was detected using the anti-type 3 capsule monoclonal antibody 16.3 (10) at a 1:10,000 dilution, as described previously (27). The amount of capsule was calculated from a standard curve generated using purified type 3 polysaccharide (American Type Culture Collection). The lower limit of detection of purified type 3 capsule was 0.01 µg/ml. Capsule production by type 2 strains was quantified in an indirect ELISA as described above, using type 2 polysaccharide-specific antiserum (Statens Seruminstitut) at a 1:20,000 dilution. C-polysaccharide (teichoic acid) was measured in an indirect ELISA as described above, using polyclonal anti-C-polysaccharide antisera (Statens Seruminstitut) at a 1:10,000 dilution.

Colonization and mouse virulence. Female 8- to 12-week-old BALB/cByJ and CBA/N (CBA/CaHN-Btk^xid) mice were used (Jackson Laboratories, Bar Harbor, Maine). Intranasal (i.n.) inoculations were performed as previously described (56), with minor changes. Briefly, a 10- to 100-ml bacterial culture was grown to a density of 3 × 10⁸ CFU/ml in THY at 37°C. The culture was centrifuged at 12,000 × g for 20 min at 4°C. The pellet was suspended in lactated Ringer's solution, and 10 µl of suspension containing 10⁷ to 10⁹ CFU of bacteria was introduced into the nares of mice. Seven days postinoculation, mice were sacrificed by asphyxiation in a CO₂ chamber. The trachea was cut at the top of the larynx, and 200 µl of lactated Ringer's solution was washed through the nares with a tuberculin syringe fitted with Intramedic Polyethylene PE20 tubing (Becton Dickinson, Sparks, Md.). A second wash with 2 ml of Ringer's solution did not yield significant numbers of additional bacteria. Serial dilutions of the nasal washes were plated on blood agar plates containing either no antibiotic, 1 µg of gentamicin/ml, 1 µg of gentamicin/ml and 10 µg of optochin (ethylhydrocupreine-HCl)/ml, or 0.3 µg of erythromycin/ml (where applicable). From these plates, the numbers of pneumococci present in the nasal washes were determined; pneumococci are capable of growth on gentamicin but not optochin. All bacteria isolated in these washes were encapsulated, as evidenced by colony morphology and confirmed by the Quellung reaction, which produces a smaller zone of reactivity with less encapsulated strains. PCR using primers specific for pneumococcal surface protein A (pspA) was used to further confirm pneumococcal identity. The primer pairs used were PspA-18 (5'-CCCAAGCTTAATATAAGTATAG-3') and PspA-11 (5'-AGGCGCGTCGA/CTCATTAACTGCTTTCTT-3') for bp 76 to 1083 or PspA-16 (5'-GTCTCAGCCTACTGTTGT-3') and PspA-11 for bp 196 to 1083. Base pair numbers correspond to the published pspA sequence (58). In some cases, lungs of i.n.-infected mice were homogenized and plated on blood agar plates to test for the presence of bacteria.

Statistics. The numbers of bacteria recovered from nasal washes were compared using an unpaired, two-tailed Student t test. The numbers of mice colonized and the numbers of mice that survived infection were each compared to those for the parent strain using a two-tailed Fisher exact test. Median times to death were compared using an unpaired, two-tailed Mann-Whitney test. Capsule production by mutant strains and that by parent strains were compared using an unpaired, two-tailed Student t test.

	RESULTS

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Capsule production. The strains used in these studies produce various amounts of capsule as a result of specific mutations in either the capsule locus or other genes known to affect capsule synthesis. Their construction and properties are described in detail in Materials and Methods and in Table 1. The amount of capsule produced by each of the strains is shown in Tables 2 and 3. Synthesis of the type 3 capsule requires a UDP-Glc dehydrogenase (UDP-Glc  UDP-glucuronic acid [GlcUA]) and the type 3 polysaccharide synthase (UDP-Glc + UDP-GlcUA  [Glc-GlcUA]_n). Both enzymes are encoded by genes (cps3D and cps3S, respectively) in the type 3 capsule locus, which is transcribed as a single operon (cps3DSUM-tnpA-plpA) (13, 19, 20). Mutants AM188, JD614, and JD692 contain different point mutations in cps3D that result in decreased capsule synthesis. Repair of each of these mutations results in restoration of parental levels of capsule (references 19 and 20 and this study). Mutants AM199 and JD908 contain insertions that result in loss of Cps3S and capsule expression. Resolution of the insertions restores parental levels of capsule production (reference 27 and data not shown). AM161 and JD770 contain insertions downstream of cps3S that do not affect capsule production (27). These strains were used as controls to ascertain any polar effects of the insertions or of the antibiotic marker (erythromycin) contained on the insertion plasmid. Cps3U, a Glc-1-P uridylyltransferase (Glc-1-P  UDP-Glc) and Cps3M, a phosphoglucomutase (PGM) homologue, are encoded within the type 3 locus but are not essential for capsule production or systemic infections with type 3 strains (13, 19, 26, 27). Strain AM179 contains a deletion of cps3UMtnpA and is not altered in capsule production (Table 2). The PGM activity necessary for conversion of Glc-6-P to Glc-1-P and synthesis of the type 3 capsule is encoded by pgm, which is found in strains of all capsule types and is unlinked to the capsule locus (26). PGM is involved in a number of other cellular pathways, including those leading to the teichoic acids. JY1060 contains a point mutation in pgm that results in decreased capsule synthesis (Table 3) and either modestly reduced virulence (in CBA/N mice) or avirulence (in BALB/cByJ mice) following systemic infection (27). Repair of the JY1060 point mutation restores parental levels of capsule and virulence (26, 27). AM1000, the capsule-negative derivative of the type 2 strain D39, was constructed by deleting the first nine genes of the D39 capsule locus (Table 3). This is the same deletion contained in R36A, which is the spontaneous, highly passaged, nonencapsulated derivative of D39 (4, 31). For all of the mutant and parent strains, no differences were detected in the teichoic acid levels, and all appeared opaque.

Nasopharyngeal colonization. The abilities of strains producing reduced levels of capsule to colonize were assessed using BALB/cByJ mice in a previously described model (56). In this model, nonanesthetized adult mice are inoculated i.n. with low-volume inocula. Under these conditions, stable colonization occurs for at least 2 weeks. The pneumococci do not cause invasive disease, and the bacteria recovered from the nasopharyngeal cavity are not the result of infections in the blood or lungs (56). In the present studies, we also did not recover bacteria from the lungs of infected mice.

Ability of the capsule-reduced mutant AM188 to cause invasive disease. Previous studies showed that, upon i.n. inoculation of CBA/N mice, A66 invades the host, resulting in systemic infection and death (56). These mice express the X-linked immunodeficient (XID) phenotype and respond poorly to polysaccharide antigens, including the S. pneumoniae capsule and the phosphocholine component of the cell wall (45, 53). Due in part to this deficiency, they are highly susceptible to pneumococcal infections (9, 10). Following i.n. inoculation of CBA/N mice, AM188 showed reduced lethality compared to that of the parent A66, and the capsule-negative AM199 was completely avirulent (Table 4). The times to death for mice that succumbed to infection with AM188 were not different from those observed with mice infected with A66 (data not shown). Mice that did not die following infection with AM188 were colonized at a frequency similar to that observed for BALB/cByJ mice (Tables 2 and 4).

	DISCUSSION

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A number of surface components have been shown to be important in colonization by S. pneumoniae. Using an established model of nasopharyngeal colonization in mice, we have shown that capsule also has an important role in this step of the infectious process. The requirement for capsule may reflect its ability to prevent clearance of the organism by innate defenses. Alternatively, or in addition, the capsule itself may be an adhesin. Either possibility is consistent with previous observations demonstrating variability among strains of different capsular serotypes in the ability to colonize, activate complement, and bind antibody to surface antigens (2, 14, 21, 29, 49, 50, 56; M. R. Abeyta and J. Yother, unpublished data). The fact that strains producing substantially reduced levels of capsule colonize as effectively as do their parent strains indicates either that the amount of capsule produced in vitro does not reflect that produced in vivo or that there is no advantage in producing excessive amounts of capsule. Indeed, we anticipate that a reduction in the amount of capsule may be a necessary step for efficient colonization, as it would allow greater exposure of surface molecules important in adherence. A reduced amount of capsule has previously been shown to correlate with enhanced colonization by transparent-phase variants, compared to opaque-phase variants, which produce elevated levels of capsule and have enhanced virulence in systemic infections (32, 50). In in vitro studies, fully encapsulated strains show reduced adherence and invasion compared to those of nonencapsulated isolates, suggesting that the capsule is an impediment to these processes (1, 17, 32, 42, 44, 48). Reduced expression of capsule by defined mutants clearly results in greater access of antibodies and complement to the pneumococcal surface (27). In the nasopharyngeal environment, appropriate signals may result in a reduction of capsule expression and an increase in expression of factors necessary for adherence. Indeed, we would expect that transmission of S. pneumoniae between carriers involves strains that are already reduced in capsule production and optimized for carriage. Hence, our type 3 mutants may remain efficient colonizers because a reduction in capsule levels is the normal scenario. Little is known about the mechanisms involved in the regulation of capsule expression. If, however, the parental mechanisms of regulation remain operative in our type 3 mutants with reduced levels of capsule production, the amount of capsule necessary for efficient colonization may well be less than that produced by AM188 and JY1060.

Previous studies demonstrated a correlation between i.p. infection virulence in mice and the level of capsule produced in vitro (32, 35). MacLeod and Krauss observed significant differences in the 50% lethal doses (LD₅₀s) of type 3 strains in which the amount of capsule varied by 2.5-fold (35). In contrast, we observed only modest differences in the times to death for A66 and AM188, which differ by fivefold in type 3 capsule production. Unlike the spontaneous isolates of MacLeod and Krauss and the phase variants of Kim and Weiser (32), our strains are the result of defined mutations. AM188 is altered only in capsule production. In contrast, JY1060 produces approximately the same amount of capsule as does AM188 but is completely avirulent in BALB/cByJ mice via the i.p. route (27). Here, the reduction in capsule is due to a mutation in pgm, and other cellular pathways are likely also affected. In addition, suppressor mutations that enhance the virulence of JY1060 do not always result in increased levels of capsule (27). Thus, reductions in capsule alone appear to have minimal effects on i.p. virulence. This is not the case for i.v. virulence, however, as both AM188 and JY1060 were significantly reduced in the ability to cause lethal infections in BALB/cByJ mice via this route (reference 27 and this study). This result may suggest that the peritoneal cavity is a relatively safe environment where bacteria do not immediately encounter the bloodstream and where a focus of infection can be maintained. Bacteria escaping this environment may then express alternative or enhanced levels of virulence factors that promote their survival in the bloodstream.

In the immunodeficient CBA/N mouse, type 3 strains inoculated i.n. cause lethal invasive infections. In contrast, AM188 was reduced in this ability but could efficiently colonize in this mouse strain. Because AM188 was highly virulent when administered i.v. in CBA/N mice, the i.n. result suggests that the capsule is either important in invasion or sufficiently reduced in quantity in AM188 by the time invasion occurs that it no longer prevents phagocytosis.

Studies with Staphylococcus aureus and Streptococcus pyogenes have also demonstrated an important role for capsule in nasopharyngeal colonization (30, 33, 52). In S. pyogenes, binding of the hyaluronic acid capsule to CD44 mediates adherence in vitro and in the nasopharyngeal cavity (40). The specific role or roles of the pneumococcal capsule in colonization are now under investigation, as are the effects of factors known or suspected to influence colonization and subsequent infections. In particular, the effects of capsular serotype, administration of anesthetics or antibiotics prior to i.n. inoculation, or preceding viral infection may alter the range of isolates that can become established in the nasopharyngeal cavity (23, 33, 38, 39, 47). Clearly, high levels of capsule expression are not required or even advantageous in all in vivo environments. An important step toward understanding the infectious process will thus be the identification of mechanisms involved in regulating capsule expression.