Molecular Pathogenesis

Two DHH Subfamily 1 Proteins Contribute to Pneumococcal Virulence and Confer Protection against Pneumococcal Disease

L. E. Cron, K. Stol, P. Burghout, S. van Selm, E. R. Simonetti, H. J. Bootsma, P. W. M. Hermans
J. N. Weiser, Editor
DOI: 10.1128/IAI.01383-10

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ABSTRACT

Streptococcus pneumoniae is an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.

INTRODUCTION

Streptococcus pneumoniae is a Gram-positive bacterium causing serious invasive diseases, such as pneumonia, septicemia, and meningitis. In addition, this human pathogen is the causative agent of less serious but highly prevalent mucosal infections, such as otitis media (OM) and sinusitis. Young children under the age of 2 years, the elderly, and immunocompromised individuals account for the majority of pneumococcal morbidity and mortality observed worldwide (10). One of the main virulence factors of the pneumococcus is its polysaccharide capsule, of which over 90 serotypes distinct in biochemical structure have been identified to date. The polysaccharide capsule contributes to protection against phagocytosis, enabling the pneumococcus to evade the immune system of the host (23, 24). Current pneumococcal vaccines, such as Pneumovax 23 (23-valent; Merck), Synflorix (10-valent; GlaxoSmithKline, United Kingdom), and Prevnar (7- and 13-valent; Pfizer) target this polysaccharide capsule. While these vaccines do provide good immune protection, it is restricted to the serotypes included in the vaccine. As a result, serotype replacement is already occurring, and the need for a serotype-independent vaccine is urgent (17, 38).

A promising alternative vaccine approach explores the use of immunogenic, surface-exposed protective proteins. Pneumococci are estimated to express over 100 surface proteins, of which some are known to have a role in pathogenesis and virulence, but mostly their function is unknown (70). Among these surface proteins that are under investigation as vaccine candidates are the pneumococcal surface proteins A and C (PspA and PspC) and the pneumococcal surface adhesin A (PsaA). The choline-binding proteins PspA and PspC are key virulence factors due to their ability to interfere with complement deposition. PspA is expressed by all pneumococci, is highly immunogenic, and is an inhibitor of C3, preventing opsonization and hence phagocytosis (59, 60, 68). PspC is a paralog of PspA which interacts with the complement-inhibitory factor H (21, 33, 48). PsaA is a metal-binding lipoprotein with specificity for Mn2+ and Zn2+ (22), both essential ions for pneumococcal growth and survival. One of the most well-studied proteins is pneumolysin (Ply). Although Ply is not a surface protein, it is an important virulence factor that is produced by all known clinical isolates of S. pneumoniae. Ply is a member of the cholesterol-dependent pore-forming toxin family that can directly damage epithelial surfaces and reduce the migration of phagocytic polymorphonuclear leukocytes (PMNs) (66). Vaccination studies using the above-mentioned proteins have shown that they can protect mice from pneumococcal disease (1, 14, 15, 45, 49–52, 69). In addition, these studies have clearly demonstrated that combinations of distinct pneumococcal proteins often enhance protection. Although these results are promising, there is still a need for additional protein targets to further improve existing experimental protein vaccine formulations.

Throughout the last decade, many genomic tools have been developed and used to identify potential protein vaccine targets, such as transcriptome analysis (53), differential fluorescence induction (43), and signature-tagged mutagenesis (STM) (27, 37, 57). We recently developed genomic array footprinting (GAF), a mutant library-based negative selection screen that uses microarrays to identify conditionally essential genes (8, 18). We have applied the GAF technology to various in vivo animal models of pneumococcal disease, i.e., colonization, bacteremia, and meningitis, to identify S. pneumoniae genes essential during infection (30, 31, 47). Interestingly, two pneumococcal proteins, SP1298 and SP2205, consisting of 311 and 657 amino acids, respectively, were consistently identified in all infection models. These two conserved proteins, of an as yet unknown function, are annotated as DHH subfamily 1 proteins in the pneumococcal genome. DHH family proteins belong to the superfamily of phosphoesterases (PE) and are named after their characteristic amino acid signature, Asp-His-His (DHH), found in a conserved N-terminal motif (i.e., motif III) (Fig. 1). The DHH family can be further divided into two distinct subfamilies, which share the four conserved N-terminal motifs but have different C-terminal motifs. Subfamily 1 proteins are found predominantly in bacteria and archaea, while subfamily 2 proteins are also represented in eukaryotes. H-prune, the human homolog of the Drosophila prune protein, is an example of a eukaryotic DHH subfamily 2 protein suggested to be involved in cell migration (20). DHH domain proteins have been identified in a variety of prokaryotes, such as Bacillus subtilis, Escherichia coli, Helicobacter pylori, Mycoplasma species, and Streptococcus gordonii (2). RecJ of E. coli is one of the most well-known bacterial DHH subfamily 1 proteins possessing exonuclease activity (42, 63). Recently, SMU.1297, a DHH-domain containing protein, was found in another streptococcal species, Streptococcus mutans (75). Yet little is known about DHH subfamily 1 proteins of S. pneumoniae.

Fig. 1.
Fig. 1.

Sequence alignment of DHH motifs from selected bacterial DHH subfamily 1 proteins. The numbers of amino acids not shown in the alignment are depicted in parentheses. Bold black shading indicates conserved residues, and gray shading indicates similarity between residues. Dashes indicate gaps created to optimize alignment within motifs. Sequences are listed in order of similarity to sequence of SP1298. SP1298, S. pneumoniae TIGR4 SP1298; SMU1297, S. mutans AU159 SMU.1297; YtqI, B. subtilis YtqI; MgORF1, M. genitalium MG_190; MpORF4, M. pneumoniae MPN140; SP2205, S. pneumoniae TIGR4 SP2205; SMU2140, S. mutans AU159 SMU.2140c; YybT, B. subtilis YtqI; Sp_RecJ, S. pneumoniae TIGR4 SP0611 (RecJ); Ec_RecJ, E. coli RecJ.

In the current study, we examined the contributions of SP1298 and SP2205 to pneumococcal virulence using single and double knockout mutants of three different strains of S. pneumoniae: D39, TIGR4, and BHN100 (serotypes 2, 4, and 19F, respectively). We chose to examine the effects of both DHH subfamily 1 proteins in three pneumococcal strains, since it has been shown that the genetic background can have a major influence on the contribution of proteins to virulence (9, 19). Using four different murine models of single and mixed infections representing major phases of pneumococcal carriage and disease, we characterized the roles of both DHH subfamily 1 proteins in the pneumococcal pathogenesis of colonization, otitis media, pneumonia, and bacteremia. We also assessed the contributions of both DHH subfamily 1 proteins to pneumococcal adherence in vitro. Finally, we provided evidence that vaccination with a combination of recombinant DHH subfamily 1 proteins can provide substantial protection against TIGR4-induced pneumonia in mice, and we propose that these proteins should be considered potential vaccine candidates.

MATERIALS AND METHODS

Pneumococcal strains and growth conditions.Pneumococcal strains used in this study are shown in Table 1. S. pneumoniae was routinely grown in Todd-Hewitt broth supplemented with 5 g liter−1 yeast extract (THY) or on Columbia blood agar (BA) plates (Oxoid) at 37°C and 5% CO2. Prior to mouse infection experiments, bacteria were passaged in mice to maintain virulence as described previously (34). Cultures of mouse-passaged S. pneumoniae strains were grown to an optical density (OD) at 620 nm (OD620) of 0.2, and aliquots were stored at −80°C in 15% glycerol. Prior to infection, defrosted aliquots were centrifuged, and bacteria were resuspended in sterile phosphate-buffered saline (PBS) to the desired concentration. When appropriate, antibiotics were used at the following concentrations: spectinomycin, 150 μg ml−1; trimethoprim, 25 μg ml−1; and kanamycin, 500 μg ml−1. The number of CFU per ml in a particular sample was quantified by plating serial 10-fold dilutions in PBS on BA plates.

View this table:
Table 1.

Bacterial strains, primers, and plasmids used in this study

Construction of site-directed deletion mutants.All primers and plasmids used in this study are shown in Table 1. A megaprimer PCR method (18) was employed to replace target genes in the genomes of the S. pneumoniae TIGR4, D39, and BHN100 strains with the spectinomycin resistance cassette amplified from plasmid pR412T7 (8). The resulting PCR products were introduced by competence-stimulating peptide (CSP)-induced transformation into the corresponding strains, using CSP-1 for D39 and BHN100 and CSP-2 for TIGR4. Transformants were selected on the basis of spectinomycin resistance and were checked by PCR for recombination at the desired location on the chromosome. In addition, a ΔSP1298 ΔSP2205 double mutant was generated in each of the three pneumococcal strains. To this end, the SP1298 gene was inactivated by allelic replacement with a trimethoprim cassette and introduced into the respective ΔSP2205 strains (spectinomycin) by transformation.

Genetic complementation of DHH mutants.Genetic revertants of the SP1298 and SP2205 BHN100 single mutants were created using CEP, a chromosomal expression platform for ectopic, maltose-driven gene expression in S. pneumoniae (25). To this end, the genes were amplified with the primer pairs HBSP1298atg/HBSP1298stop and HBSP2205atg/HBSP2205stop, respectively, using BHN100 chromosomal DNA as a template. After digestion by NcoI/BamHI, the SP1298 and SP2205 fragments were ligated with the NcoI/BamHI-digested plasmid pCEP. The resulting SP1298 and SP2205 ligation mixtures were used as the donor in transformation of strains BHN100ΔSP1298 and BHN100ΔSP2205, respectively, followed by selection for kanamycin-resistant transformants, thus generating the strains BHN100ΔSP1298 CEPSP1298 and BHN100ΔSP2205 CEPSP2205. The complemented mutants were checked by PCR for integration at the desired location on the chromosome. A control CEPØ strain lacking a gene insert was constructed by transforming BHN100 wild type with HindIII-digested pCEP and selecting for kanamycin-resistant transformants. To examine SP1298 and SP2205 gene and protein expression levels, bacterial strains were grown to mid-log phase in THY medium without or with the addition of 0.4% maltose. Aliquots of these cultures were stored at −80°C with 15% glycerol for adherence assays.

Real-time PCR.Total RNA was extracted using the RNeasy minikit (Qiagen), after which contaminating genomic DNA was removed by treatment with DNase (DNAfree; Ambion). DNA-free total RNA (2.5 μg) was reverse transcribed using 300 ng of random hexamers and Superscript III reverse transcriptase (Invitrogen). To confirm the absence of genomic DNA, reactions without reverse transcriptase were carried out. Relative amounts of SP1298 and SP2205 transcripts were determined by quantitative real-time-PCR (qRT-PCR) using the SYBR green technology on a 7500 Fast real-time PCR system (PE Applied Biosystems) according to the manufacturer's instructions. The relative quantification method was used to evaluate the quantitative variation between wild-type and complemented strains for each gene examined (39). The gyrA (SP1219) amplicon was used as an internal control for normalization of data.

Production of His-tagged SP1298 and SP2205 and generation of polyclonal rabbit antisera.The SP1298 and SP2205 genes of S. pneumoniae TIGR4 were PCR amplified with the oligonucleotide primer pairs LCSP1298AvrH6F/LCSP1298BamR and LCSP2205AvrH6F/LCSP2205BamR, respectively. The amplicons were cloned into the pCR2.1 cloning vector of the TA cloning kit (Invitrogen) to obtain pLC1a (SP1298) and pLC2 (SP2205). Since sequence analysis showed that the AvrII site was not intact for SP1298, we PCR amplified SP1298 from pLC1a using the primer pair LCSP1298XbaF/LCSP1298BamR and subcloned the amplicon into pCR2.1 to obtain pLC1b. In the next step, the recombinant genes were excised with either XbaI/BamHI (SP1298) or AvrII/BamHI (SP2205) digestion and ligated to the BamHI/NheI-digested pET11c expression vector to obtain pLC1298 and pLC2205, respectively. The nucleotide sequences of the SP1298 and SP2205 genes in pLC1298 and pLC2205 were confirmed by sequence analysis.

For the production of His-tagged SP1298 (rHisSP1298) and His-tagged SP2205 (rHisSP2205), an overnight culture of E. coli BL21 (pLC1298 or pLC2205) was diluted 50-fold in prewarmed (37°C) 2× LB supplemented with 0.5% glucose and 100 μg ml−1 ampicillin. At an OD600 between 0.6 and 0.8, 0.1 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) was added to the culture. After 2 h, cells were placed on ice, pelleted by centrifugation, resuspended in ice-cold lysis buffer consisting of 20 mM sodium phosphate, 0.5 M sodium chloride, and 10 mM imidazole (pH 7.4) to an equivalent of an OD600 of 100, and lysed by sonication. For pLC2205, 6 M urea, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1% Triton X-100 were added to the lysis buffer. For complete cell lysis of the pLC2205 culture, 6 M urea, 1 mM PMSF, and 0.1% Triton X-100 were first added to the lysis buffer and the suspension was frozen at −20°C. Subsequently, a defrosted cell suspension of pLC2205 was sonicated in the presence of 100 mM PMSF, 100 mM benzamidine (BZA), and lysozyme (100 mg ml−1). Insoluble debris in both lysates was removed by ultracentrifugation (Sorvall WX ultracentrifuge in a Ti70.1 Beckmann rotor) at 40,000 rpm for 1 h at 4°C. The resulting supernatants were loaded onto a 1-ml HiTrap Chelating HP column (Amersham Biosciences) preloaded with Ni2+, washed with 10 mM imidazole phosphate buffer, and eluted with 300 mM imidazole phosphate buffer, and the fractions containing purified rHisSP1298 or rHisSP2205 were combined and dialyzed (Slide-A-Lyzer dialysis cassette 3500 MWO, 0.5 to 3 ml capacity; Pierce). Dialysis buffer for rHisSP1298 contained 10 mM HEPES, while rHisSP2205 was dialyzed against 10 mM HEPES, 6 M urea, and 0.1% Triton X-100. After dialysis, rHisSP1298 was lyophilized and rHisSP2205 was stored in the dialysis buffer; both were stored at −20°C until further use. The identities of the purified proteins were confirmed by matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) analysis, and their amounts were determined by the bicinchoninic acid (BCA) assay (Bio-Rad).

For the generation of anti-SP1298 and anti-SP2205 polyclonal rabbit antibodies, a total of 400 μg of each protein was used according to the Speedy program of Eurogentec.

Western blot analysis.Whole-cell bacterial lysates were used in Western blot analysis by separating 2.5 × 107 CFU/lane on SDS-PAGE gels (12.5%). After electrophoretic transfer onto a nitrocellulose membrane, transfer was visualized by staining membranes with Ponceau S (Sigma-Aldrich). Subsequently, membranes were blocked using PBS with 0.1% Tween 20, 2% skim milk powder, and 1% bovine serum albumin (BSA) and incubated with the primary antibodies (rabbit polyclonal anti-SP1298 or anti-SP2205) followed by horseradish peroxidase-coupled secondary anti-rabbit antibody. Immunoblots were developed by enhanced chemiluminescence (Amersham).

Pneumococcal adherence assay.The human pharyngeal epithelial cell line Detroit 562 (ATCC CCL-138) was routinely grown in RPMI 1640 medium without phenol red (Invitrogen, The Netherlands) supplemented with 1 mM sodium pyruvate and 10% (vol/vol) fetal calf serum (FCS). All cells were cultured at 37°C in a 5% CO2 environment. For adherence assays, bacteria were resuspended in RPMI 1640 medium without phenol red supplemented with 1% FCS. For adherence experiments with complemented mutants, maltose was added to the medium to a concentration of 0.4%. Adherence of pneumococci to epithelial cells was carried out as described previously (11, 19). Briefly, Detroit 562 cells were seeded into 24-well plates and incubated for 48 h. Confluent monolayers were washed twice with 1 ml PBS and infected with 1 × 107 CFU ml−1 (multiplicity of infection [MOI] of 10 [bacteria/cells]), and pneumococci were allowed to adhere to the cells for 2 h at 37°C in a 5% CO2 environment. Nonadherent bacteria were removed by 3 washes of 1 ml PBS, after which 200 μl of 0.05% trypsin–1 mM EDTA was added to detach the cells, followed by 800 μl of ice-cold 0.025% Triton X-100 in PBS to lyse the cells. Samples were plated for CFU count and corrected to account for small differences in the initial inoculum. All experiments were performed in triplicate and repeated at least three times. The adherence of the mutants is given as the percentage relative to results for the wild type. All bacterial strains grew equally well in the tissue culture medium.

Mouse studies.Eight-week-old female outbred CD-1 mice (Charles River Laboratories, Germany) were used for the colonization, pneumonia, and bacteremia models, while the OM experiments were performed with 7-week-old female inbred specific-pathogen-free BALB/c mice (Harlan, The Netherlands). All mice were housed in filter-top cages and had access to food and water ad libitum. Mice were allowed to acclimate for a week prior to each experiment. All animal experiments were performed with approval of the Radboud University Nijmegen Medical Centre Committee for Animal Ethics.

Pneumonia model.Mice were lightly anesthetized with 2.5% (vol/vol) isoflurane over oxygen and infected intranasally (i.n.) by pipetting 50 μl of inoculum (5 × 106 CFU total) into the nostrils of mice while they were held in an upright position. At predetermined times after infection, groups of mice were sacrificed by injection anesthesia, and blood samples were taken by retro-orbital bleeding. Bacteria were recovered from the nasopharynx by flushing each nare with 1 ml sterile PBS (nasopharyngeal lavage [NPL]) (28). A bronchoalveolar lavage (BAL) was performed by flushing the lungs with 2 ml sterile PBS, after which lungs were removed from the body and homogenized in 2 ml sterile PBS using a handheld homogenizer (Polytron PT 1200; Kinematica AG).

Colonization model.Mice were infected i.n. under anesthesia as mentioned above with a smaller volume of inoculum, 10 μl (5 × 106 CFU total). As described previously, nasal instillation of pneumococci with such a low volume does not cause a lethal infection in mice (19). Mice showed no visible signs of disease throughout the course of colonization and had less than 40 CFU in the lungs at the last time point. At predetermined time points after infection, NPL and lungs were collected as described above.

Otitis media model.Intranasal infection was performed as described above in the colonization model with the following exception: methylcellulose (1%) was added to the inoculum for all OM experiments in order to minimize leakage of inoculum to the lungs (67). Mice were placed in a supine position in the pressure cabin after infection as described previously (62). Briefly, an initial pressure rise was set at 10 kPa, and when the mouse started to regain consciousness and the first swallowing movements occurred, pressure was raised at the rate of 5 kPa per 15 s until a pressure of 40 kPa was reached, enabling the inoculum to reach the middle ear cavity. Then, the pressure was lowered gradually until atmospheric pressure was reached again. Groups of mice were sacrificed at 24, 48, and 96 h postinfection, where mice were bled out by retro-orbital puncture, followed by cervical dislocation. The bullas enclosing the middle ears (ME) were dissected from the temporal bone and homogenized in the presence of 1 ml sterile PBS per ear, as previously described (62, 67). Bacteria were also recovered from the nasopharynx by performing an NPL using 1 ml of sterile PBS, and the lungs were extracted and homogenized in 2 ml sterile PBS.

Bacteremia model.Mice were infected via the tail vein with a 100-μl inoculum (106 to 107 CFU total). To confirm successful infection, blood was taken from a separate vein immediately after injection (t = 0 h). Subsequently, blood was recovered via a tail vein puncture from the same mouse at 12 and 24 h postinfection and by retro-orbital puncture at the last time point, 36 h.

Coinfection experiments.A 1:1 ratio of the wild type and its respective ΔSP1298, ΔSP2205, or ΔSP1298 ΔSP2205 mutant was used to infect the mice as described for the above-mentioned infection models. This setup reduces variation between individual mice, inoculation preparation and distribution, and sample collection. Viable bacteria were quantified by plating serial dilutions on BA plates, and BA plates supplemented with either spectinomycin or spectinomycin and trimethoprim. Subsequently, competitive index (CI) scores were calculated for each individual animal as the output ratio of mutant to wild-type bacteria divided by the input ratio of mutant to wild-type bacteria. A log CI score of <0 indicates that the mutant is outcompeted by the wild type. For samples in which no viable mutant bacteria were recovered, the lower limit of detection (20 CFU ml−1) was substituted as the numerator. If in one particular sample neither wild-type nor mutant bacteria were detected, the data were excluded from further analysis.

Immunizations.Female CD-1 mice (6 weeks old) were subcutaneously immunized three times at 14-day intervals with a total of 50 μg protein in alum adjuvant (aluminum hydroxide gel; Sigma). Briefly, mice were either primed singly with rHisSP1298 or rHisSP2205 or with a combination of the two proteins. The negative-control group consisted of mice given a 1:1 ratio of alum adjuvant and PBS. One-tenth of the human dose of Prevnar 7 was given to the positive-control group. Blood samples from all mice were collected via tail vein puncture prior to any immunization, at the time of the third boost, and several days before infection. Mice were subsequently challenged i.n. with the TIGR4 wild-type strain (1 × 106 CFU total) 3 weeks after the last immunization in our pneumonia model and sampled 48 h postinfection as described above.

Detection of antigen-specific IgG by ELISA.IgG titers against SP1298 and SP2205 were determined by enzyme-linked immunosorbent assay (ELISA) analysis. High-binding-capacity microtiter plates (Greiner) were coated with 1 μg μl−1 purified rHisSP1298 or rHisSP2205 in 100 μl per well overnight at 4°C. Plates were washed with PBS with 0.05% Tween 20 (PBST) and then incubated for 1 h with PBST containing 2% BSA. Three-fold serial dilutions of sera were added to the plates and incubated for 1 h at 37°C. After washing, the alkaline phosphatase secondary antibody directed to mouse IgG-Fc (Sigma-Aldrich) was added for 1 h at 37°C using a 1:25,000 dilution. After washing, 100 μl per well of p-nitrophenyl phosphate (1 mg ml−1) in substrate buffer (10 mM diethanolamine and 0.5 mM magnesium chloride, pH 9.5) was added and the absorbance was read at 405 nm.

Statistical analyses.For adherence assays, comparisons between wild-type and mutant pneumococcal strains were performed using Student's t test (unpaired). The Mann-Whitney test was used for comparison of bacterial load in NPL, ME, BAL fluid, lung homogenate, and blood between the wild-type-infected mice and their respective DHH mutant-infected mice in all infection models. For the coinfection data, a Wilcoxon test on log-transformed CI scores was used to determine if the median CI was statistically significantly different from 0 (i.e., no outcompetition). All statistical analyses were performed using the GraphPad Prism software program, version 4.0.

RESULTS

DHH subfamily 1 proteins contribute to pneumococcal adherence in vitro.To assess the (strain-specific) contribution of the DHH subfamily 1 proteins SP1298 and SP2205 to pneumococcal pathogenesis, directed mutants were generated in three strains: D39, TIGR4, and BHN100. Western blot analysis of wild-type, ΔSP1298, ΔSP2205, and double mutant cell lysates using anti-SP1298 and anti-SP2205 rabbit serum confirmed that SP1298 and SP2205 were expressed in all three wild-type strains but not in their respective single and double mutants (data not shown). Importantly, no differences in in vitro growth between the wild-type strains and the DHH subfamily 1 mutants were detected.

Adherence of pneumococci to respiratory epithelial cells is crucial for colonization of the nasopharynx. Therefore, we examined adherence of the three wild-type strains and their isogenic single and double DHH subfamily 1 mutants to human pharyngeal epithelial Detroit 562 cells. Wild-type adherence levels of TIGR4 (∼6.5 × 104 adherent CFU) were statistically significantly higher than those of the wild types of the D39 and BHN100 strains by almost 1 log. Interestingly, no difference in adherence was observed upon deletion of the SP1298 gene in TIGR4 and D39, while adherence of BHN100ΔSP1298 was reduced by ∼75%, suggesting that the contribution of SP1298 to adherence is strain specific (Fig. 2 A). In contrast, all three SP2205 mutants showed a significant decrease in adherence compared to their wild types, ranging from 60 to 90% (Fig. 2A). The ability of the double mutants in all three strains to adhere was drastically reduced, by >80%, displaying an enhanced effect when both genes were deleted (Fig. 2A).

Fig. 2.
Fig. 2.

(A) In vitro adherence of pneumococcal strains to the human pharyngeal epithelial cell line Detroit 562. The adherence of the mutants is given as the percentage relative to that for the respective wild type. (B) Real-time PCR (upper panels) and Western blot analysis (lower panels) of BHN100 complemented SP1298 (left) or SP2205 (right) mutants. Real-time data are expressed as the log2 ratio of expression relative to that for the BHN100 wild type grown without maltose. Strain names and presence of maltose in growth medium are indicated at the bottom. (C) In vitro adherence of BHN100 complemented mutants to Detroit 562 cells, given as the percentage relative to that for the BHN100 wild type grown without maltose. The presence of maltose in growth medium is indicated at the bottom. All values are geometric means, and error bars represent SEM. *, P < 0.0001.

To confirm that the observed phenotypes were indeed due to the deletion of the SP1298 and SP2205 genes, we generated genetically complemented mutants by ectopic expression of the respective genes from the maltose-inducible CEP site. Given that attenuated adherence of both single mutants was observed only in the BHN100 strain, we chose this background for genetic complementation. As a control, we generated BHN100 CEPØ, containing the empty CEP platform. Real-time PCR and Western blot analysis of the genetic revertants showed that SP1298 gene and protein expression was restored to wild-type levels in normal medium and was 5-fold higher than that of the wild type upon addition of maltose (Fig. 2B). For SP2205, both gene expression and protein expression were clearly restored in normal medium, but maltose induction was required to reach wild-type levels (Fig. 2B). Importantly, the ability of the complemented SP1298 mutant to adhere to Detroit cells was indistinguishable from that of the wild type when cells were grown without maltose (Fig. 2C), while the maltose-induced overexpression of SP1298 had a slight adverse effect on the adherence ability. Moreover, the adherence of the SP2205 mutant also was no longer significantly different from that of the wild type upon complementation, with or without maltose induction. In all cases, BHN100 CEPØ behaved as did the wild type. Thus, the observed attenuated adherence phenotypes of the DHH mutants were not the result of an inadvertent mutation in a nontargeted gene.

DHH subfamily 1 proteins contribute to various stages of pneumococcal pathogenesis in vivo. (i) Colonization.Since colonization of the nasopharynx is a prerequisite for pneumococcal infection, we first compared all single and double SP1298 and SP2205 mutants to their isogenic parental wild types following intranasal infection in our established colonization model. All wild-type strains maintained an overall colonization level of approximately 105 CFU during the course of infection (Fig. 3 A, C, and E). Even though all single and double SP1298 and SP2205 mutants were capable of colonizing the murine nasopharynx to various degrees throughout infection, significant decreases in bacterial load were observed compared to those of their respective wild types over time. Interestingly, SP1298 and SP2205 mutants in a BHN100 background maintained a slightly higher level of colonization in the nasopharynx than the SP1298 and SP2205 mutants in the other two pneumococcal backgrounds. The most prominent phenotype was observed in the TIGR4 genetic background, where all single and double SP1298 and SP2205 mutants were significantly attenuated at all time points from 24 h postinfection onwards (Fig. 3A). The double mutants in all three strain backgrounds were significantly impaired in their ability to colonize the nasopharynx, but again the most severe attenuation was observed in the TIGR4 background, with a 2,500-fold decrease compared to results for the wild type after just 24 h (P < 0.005) (Fig. 3A). Since both single mutants in TIGR4 showed a decrease of only ∼10-fold, this suggests an additive effect when both genes are absent. Mice infected with single and double SP1298 and SP2205 mutants of D39 (Fig. 3C) and BHN100 (Fig. 3E) also had lower bacterial loads than their respective wild-type-infected mice, especially at the later time points.

Fig. 3.
Fig. 3.

Bacterial load in the nasal lavage fluid of mice intranasally infected with 5 × 106 CFU of the wild type and/or the respective ΔSP1298, ΔSP2205, or ΔSP1298 ΔSP2205 mutants. Strain data are depicted for TIGR4 (A and B), D39 (C and D), or BHN100 (E and F). Data from single-infection experiments are shown in panels A, C, and E; data from coinfection experiments are shown in panels B, D, and F. The horizontal line represents the lower limit of detection, and error bars represent SEM. Each point in panels B, D, and F represents the log competitive index score from an individual mouse. Values < 0 indicate attenuation of the mutant. Horizontal lines represent the mean. *, P < 0.05.

To further characterize the potential role of these two DHH subfamily 1 proteins in pneumococcal colonization, we examined the phenotype of each mutant when in direct competition with its respective wild type, since minor differences between two bacterial strains can be unmasked with such a competitive setup. The TIGR4 and BHN100 wild types significantly outcompeted their respective single SP1298 and SP2205 mutants 96 h postinfection (900- to 7,000-fold; Fig. 3B and F). Interestingly, although outcompetition of the D39 single mutants was significant but much less prominent (<10-fold; Fig. 3D) than that for the other two strain backgrounds, the double mutants in all strains were outcompeted ∼500- to 7,000-fold, suggestive of a strain-specific additive effect (Fig. 3B, D, and F). Especially for the TIGR4 strain, attenuation levels of mutants differed between single infection and coinfection: both single mutants were outcompeted to the same extent as the double mutant in coinfection, whereas a 2-log difference in colonization levels was observed in single infection. This is most likely due to the enhanced sensitivity of the competitive setup, where both single mutants were already outcompeted to levels below our detection limit (20 CFU ml−1).

(ii) Otitis media.The pathogenesis of pneumococcal OM involves translocation of the bacteria from the nasopharynx to the middle ear cavity through the Eustachian tube, accomplished by the use of a pressure cabin in the murine OM model recently described (62). We examined the contribution of the DHH subfamily 1 proteins to experimental OM using the mutants in TIGR4 and BHN100 since it has been established that these two strains produce higher CFU counts in the middle ear in this model than D39 (62). Colonization levels of the middle ear with BHN100 wild type (>105 CFU ml−1) were higher than those with the TIGR4 wild-type strain (∼104 CFU ml−1) throughout the course of OM infection (Fig. 4 A and C).

Fig. 4.
Fig. 4.

Bacterial load in the middle ear fluid of mice intranasally infected with 1 × 106 CFU of the wild type and/or respective ΔSP1298, ΔSP2205, or ΔSP1298 ΔSP2205 mutants. Strain data are depicted in panels A and B for BHN100 and in C and D for TIGR4. The single infections shown in panels A and C represent the sum of pneumococci in the left and right middle ear fluid. Horizontal line represents the lower limit of detection, and error bars represent SEM. Coinfection data are shown in panels B and D, where each point represents the log competitive index score from one mouse ear. Values < 0 indicate attenuation of the mutant. Horizontal lines represent the mean. *, P < 0.05.

Attenuation of the BHN100 double mutant was observed at all time points postinfection (Fig. 4A). Reduced bacterial loads of <103 CFU ml−1 were observed for BHN100ΔSP1298 throughout OM infection, albeit only statistically significant at the last two time points, while BHN100ΔSP2205 only appeared to be attenuated at 48 h (Fig. 4A). In line with the BHN100 data, the TIGR4 double mutant was incapable of colonizing the middle ear at any of the time points (Fig. 4C). A consistent reduction in the bacterial load was observed for TIGR4ΔSP1298 and to a lesser extent TIGR4ΔSP2205 (Fig. 4C), but this was not statistically significant except for TIGR4ΔSP1298 at 96 h due to a large spread of the wild type.

In the coinfection experiments, both wild types outcompeted their respective single and double SP1298 and SP2205 mutants in the middle ear by 20- to 350-fold (Fig. 4B and D). These observations clearly confirmed the single-infection data. Finally, nasopharyngeal colonization levels of the wild type and mutants in the OM model were similar to NPL data obtained with the colonization model (data not shown).

(iii) Pneumonia.Pneumococcal pneumonia can occur when bacteria are aspirated from the nasopharyngeal niche to the lungs. In the murine pneumonia model, infection was monitored at three sites, nasopharynx, lungs, and blood, allowing us to examine aspects of both colonization and invasive disease. Nasopharyngeal colonization in the pneumonia model was comparable to the results observed in the colonization and OM models (data not shown). Interestingly, similar to our observations in the NPL (Fig. 3E), the number of BHN100 wild-type bacteria in the BAL fluid (∼104 CFU) was higher than those of the TIGR4 and D39 wild-type strains after 24 h postinfection (Fig. 5 B, E, and H). A very pronounced phenotype was seen with the BHN100 single and double SP1298 and SP2205 mutants, since they were all significantly attenuated, ∼2 logs in the lungs and BAL fluid, 24 and 48 h postinfection compared to results for the BHN100 wild type (Fig. 5G and H). In the lung tissue, we observed that the D39 double mutant was significantly attenuated at all time points, while only D39ΔSP1298 was attenuated at 48 h (Fig. 5D). However, bacterial loads for all D39 single and double SP1298 and SP2205 mutants recovered in BAL fluid at 24 and 48 h were significantly lower than wild-type loads (Fig. 5E). For TIGR4, the single ΔSP1298 mutant and the double mutant were also unable to cause infection in the lungs and BAL fluid at 24 and 48 h (Fig. 5A and B), albeit this was not always statistically significant due to a large spread for the wild type. However, infection of mice with TIGR4ΔSP2205 was capable of causing disease at wild-type levels 48 h postinfection (Fig. 5A and B).

Fig. 5.
Fig. 5.

Bacterial load in the lung homogenate and bronchoalveolar lavage fluid of mice following intranasal infection with 5 × 106 CFU of the wild type and/or respective ΔSP1298, ΔSP2205, or ΔSP1298 ΔSP2205 mutants. Strain data for TIGR4 (A, B, and C), D39 (D, E, and F), or BHN100 (G, H, and I) are shown. Data from single-infection experiments are shown in panels A, B, D, E, G, and H, and data from coinfection experiments are shown in panels C, F, and I. The horizontal line represents the lower limit of detection, and error bars represent SEM. Each point depicted in panels C, F, and I indicates the log competitive index score from an individual mouse. Values < 0 indicate attenuation of the mutant. Horizontal lines represent the mean. *, P < 0.05.

In the pneumonia coinfections, we observed that the TIGR4 and BHN100 wild types significantly outcompeted their respective SP1298 (20- to 2,200-fold) and SP2205 (70- to 10,000-fold) mutants at 48 h postinfection in the lung and BAL fluid (Fig. 5C and I), while the D39 wild type significantly outcompeted the ΔSP2205 (200-fold) and ΔSP1298 ΔSP2205 (50,000-fold) mutants only in the lung tissue (Fig. 5F). Moreover, we observed that the TIGR4 and D39 wild types outcompeted their respective double mutants much more than their respective single DHH subfamily 1 mutants, displaying an enhanced effect when both genes were deleted (Fig. 5C and F). For example, the TIGR4 double mutant was outcompeted by its wild type 40,000-fold, while the single SP1298 and SP2205 mutants were outcompeted by only 2,200-fold and 10,000-fold, respectively.

The TIGR4 and D39 strains and their respective mutants were all able to disseminate from the lungs to blood, except for the SP1298 mutants, for which no bacteria were detected at any time point (see Fig. S1 in the supplemental material). Once in the blood, the TIGR4 and D39 wild types and their respective ΔSP2205 mutants showed growth characteristics similar to those described below for the bacteremia model.

(iv) Bacteremia.Bacteremia is a severe complication which occurs in approximately 30% of pneumonia cases (12). To assess whether the DHH subfamily 1 proteins are required for survival of pneumococci once they have entered the bloodstream, we intravenously infected mice with wild-type and/or mutant pneumococci. Both wild types (TIGR4 and D39) had higher bacterial counts (7- to 25,000-fold more) in the blood than their respective SP1298 and SP2205 mutants throughout the course of the infection (Fig. 6 A and C), except for the TIGR4ΔSP2205 mutant. The fact that the TIGR4ΔSP2205 mutant behaved as did the wild-type in this model is supported by corresponding blood data from the pneumonia model. The mice infected with TIGR4ΔSP1298 maintained significantly lower levels (∼200-fold less) of bacteria after 12 h postinfection. In the case of D39, the single ΔSP1298 and double mutants were significantly attenuated at all time points, but the ΔSP2205 mutant was attenuated only at 32 h postinfection (Fig. 6C). The BHN100 strain was not capable of surviving in the blood and was cleared from the bloodstream within 24 h after infection (data not shown). Furthermore, we observed that the TIGR4 and D39 wild types statistically outcompeted their respective double mutants up to 3 logs more than their respective single DHH subfamily 1 mutants, further signifying an additive effect when both genes were deleted (Fig. 6B and D). Interestingly, the TIGR4ΔSP2205 mutant was attenuated in blood only in a coinfection setup, suggesting this mutant is able to efficiently survive during bacteremia only when it is not in direct competition with the wild type.

Fig. 6.
Fig. 6.

Bacterial load in the blood of mice over time following intravenous infection with 1 × 106/7 CFU of wild-type and/or respective ΔSP1298, ΔSP2205, or ΔSP1298Δ SP2205 mutants. Strain data for TIGR4 (A and B) or D39 (C and D) are depicted. Data from single-infection experiments are shown in panels A and C, and data from coinfection experiments are shown in B and D. The dotted horizontal line represents the lower limit of detection, and error bars represent SEM. Each point depicted in panels B and D indicates the log competitive index score from an individual mouse. Values < 0 indicate attenuation of the mutant. Horizontal lines represent the mean. *, P < 0.05.

DHH subfamily 1 proteins confer protection against pneumococcal disease.To evaluate the protection elicited by immunization with the recombinant rHisSP1298 and rHisSP2205 proteins either singly or in combination, CD-1 mice were immunized with these proteins and subsequently challenged with S. pneumoniae TIGR4. The individual IgG titers in sera of mice immunized with one or both DHH subfamily 1 proteins or with Prevnar 7 were tested 3 weeks after the final immunization. Analysis showed antigen-specific antibody responses that were significantly higher than those of their respective negative-pool preimmune serum (data not shown). No difference in IgG titers was seen between the singly and doubly vaccinated groups. At 48 h postinfection, protection was evaluated by quantifying the bacterial load. As expected, mice that received Prevnar 7 were protected (Fig. 7), since capsular type 4 is included in this vaccine. While a minor reduction of bacteria in the nasopharynx (P < 0.013) of rHisSP1298-vaccinated mice was observed (Fig. 7A), mice receiving either rHisSP1298 or rHisSP2205 alone were not protected and succumbed to infection just as rapidly as mice receiving the adjuvant only. Interestingly, protection was seen only with mice who received a combination of both DHH subfamily 1 antigens. Bacterial loads in the nasopharynx, blood, and lungs of these mice were statistically significantly lower (P < 0.0001, 0.013, and 0.003, respectively) than those of mice receiving adjuvant only (Fig. 7A, B, and D) after subsequent challenge with the TIGR4 wild type. Sixty percent of these mice did not develop bacteremia, and clinical signs of disease were minimal.

Fig. 7.
Fig. 7.

Protection against intranasal challenge with S. pneumoniae TIGR4. Mice subcutaneously immunized with alum (open squares), rHisSP1298 (filled triangles), rHisSP2205 (filled circles), a combination of rHisSP1298 and rHisSP2205 (filled diamonds), or Prevnar 7 (filled squares) were subsequently challenged with TIGR4 wild type. Pneumococci were recovered 48 h postinfection from NPL (A), blood (B), BAL fluid (C), or lung homogenate (D). Each symbol represents one mouse. The dotted horizontal line represents the lower limit of detection. *, P < 0.05.

DISCUSSION

Current polysaccharide conjugate vaccines targeting the respiratory tract pathogen S. pneumoniae provide excellent protection against invasive disease caused by vaccine serotypes yet remain ineffective against circulating nonvaccine serotypes. Vaccines that include conserved (surface) proteins involved in pneumococcal virulence are considered promising alternatives. To identify such targets, we previously screened for genes essential during pneumococcal infection using the GAF technology (8, 18, 47). These in vivo GAF screens have led to the identification of the DHH subfamily 1 proteins SP1298 and SP2205. These two proteins have not been identified by previous STM or expression studies and have consequently never been considered as virulence factors or vaccine leads in S. pneumoniae. This prompted us to examine their role in pneumococcal pathogenesis and protection in more detail.

Our in vitro and in vivo results clearly demonstrate that the two pneumococcal DHH subfamily 1 proteins are required for full pneumococcal virulence at several target sites of the host, e.g., nasopharynx, middle ear, lungs, and blood. Genetic complementation of the SP1298 and SP2205 genes in the BHN100 single mutants resulted in restoration of in vitro adherence to wild-type levels, indicating that our observed phenotypes are indeed the results of the gene deletions and not an inadvertent mutation in a nontargeted gene. Interestingly, an additive effect was observed in vitro and in vivo when both genes were deleted. These data suggest that SP1298 and SP2205 may functionally complement each other, rendering S. pneumoniae incapable of causing disease when both genes are absent. Additive or even synergistic reduction in virulence of pneumococcal mutants lacking multiple genes of complementary function has been described previously. For example, during their investigation of two iron ABC transporter systems, PiuA and PiaA, Brown et al. demonstrated that a single deletion of piuA or piaA resulted in only a moderate reduction in virulence, whereas a mutant strain lacking both genes displayed severe attenuation in both pulmonary and systemic models of infection (16). In another study, individual ΔaliA, ΔaliB, and ΔamiA mutants of the Ami-AliA/AliB oligopeptide permease were only moderately impaired in nasopharyngeal colonization, while the triple knockout obl mutant was severely attenuated (34).

Since the pneumococcal genetic background can have a significant impact on the contributions of individual genes to virulence, we performed our study using three genetically distinct strains. Adherence data showed that the ΔSP2205 and double mutants were severely reduced in adherence to Detroit 562 cells in all three genetic backgrounds compared to their respective wild types, whereas the ΔSP1298 mutant showed diminished adherence only in a BHN100 background. Conversely, in vivo data for all infection models demonstrated that the ΔSP1298 and double mutants in all three strain backgrounds were significantly attenuated, while the TIGR4ΔSP2205 mutant displayed wild-type levels of virulence in lungs and blood in single infection. Attenuation of TIGR4ΔSP2205 was observed only in coinfection experiments, specifically in the pneumonia and bacteremia models. These data suggest that not only strain-specific but also host site-specific features of strains and their respective mutants can occur. An obvious explanation for strain-specific phenotypes of mutants is the presence or absence of other virulence genes (in)directly contributing to observed phenotypes. For instance, the type I pilus that has been described to be involved in pneumococcal adherence and colonization (3) is encoded by the genomes of the TIGR4 and BHN100 strains but not by that of D39. It does not appear to be responsible for the strain-specific adherence observed in our study, though, since the D39 and TIGR4 mutants showed similar phenotypes. Strain specificity has also been described for virulence factors present in all strains; for example, the contribution of PspC to pneumococcal virulence has been shown to vary between strains, both at the level of virulence during pneumonia and bacteremia and at the level of factor H binding (35, 74), and we have demonstrated that the putative proteinase maturation protein A (PpmA) contributes to adherence in a strain-specific manner (19). Finally, transcriptional response regulators (RR), such as RR04 and RR09, have also been shown to affect both virulence and gene expression in a strain-specific manner (9, 29, 44).

A reasonable explanation for the observed phenotypes of the DHH subfamily 1 mutants may be associated with the cellular localization of the DHH subfamily 1 proteins. SP2205 is predicted to be surface exposed by virtue of N-terminal transmembrane helices, which suggests that its observed contribution to adherence may well be a direct effect, since the protein may act as an adhesin. A significant reduction in invasive disease was attributed to the deletion of SP1298, which may be the result of an indirect effect, since this protein is predicted to be cytoplasmic. SP1298 does not contain known signal sequences or typical motifs required for membrane anchoring. However, the presence of known export signals is not necessarily a prerequisite for surface exposure, as exemplified by two other pneumococcal virulence factors: the plasmin(ogen) binding proteins enolase (Eno) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (5, 54). Binding of human plasmin(ogen) by pneumococcal Eno and GAPDH enhances bacterial virulence by capturing surface-associated proteolytic activity, thus promoting penetration of bacteria through reconstituted basement membranes (5–7). Even though these proteins are predicted to be cytoplasmic based on their amino acid sequence, immunoelectron microscopy and immunoblot analysis have clearly showed that both of these glycolytic enzymes are present in the cytoplasm as well as on the bacterial cell surface (5, 6). Ply is also a signal peptide-lacking protein localized in the cytoplasm that, in addition to formation of pores, can activate complement and stimulate host cell apoptosis once released from the bacterium (13, 55, 56). Interestingly, unlike the SP1298 and SP2205 mutants, a ply-negative mutant could be complemented by the presence of the wild-type strain upon coinfection (4). This suggests that while Ply acts at a distance from the pneumococcus, the DHH subfamily 1 proteins exert their effects in connection with or very close to the cell. Whether the cellular localization of SP1298 and SP2205 is the cell surface, cytoplasm, or both remains to be investigated, since preliminary fluorescence-activated cell sorter (FACS) analysis using the polyclonal antisera was inconclusive (data not shown).

Even though the exact function of SP1298 and SP2205 in S. pneumoniae is unknown at present, their DHH domain(s) (Fig. 1) may shed some light on their putative role in pneumococcal pathogenesis. The N-terminal motifs I to IV of DHH family proteins contain the residues required for core catalytic activity, and the C-terminal, subfamily-specific motifs contribute to substrate specificity (2). In addition to the N-terminal DHH motifs, both SP1298 and SP2205 possess a DHHA1 domain, characteristic of members of DHH subfamily 1. These domains are ∼60 residues long and contain a conserved GGG motif located near the C terminus (2). DHH domain proteins are known to function as phosphatases or phospho(di)esterases (PDE) capable of hydrolyzing a wide variety of substrates from inorganic pyrophosphate to single-stranded DNA (2). A well-studied bacterial example is RecJ of E. coli, an exonuclease involved in DNA repair and recombination systems (73), of which homologues are found in S. pneumoniae (Fig. 1) and in Bordetella pertussis, Haemophilus influenzae, and Neisseria meningitidis (2, 63). Some less-well-characterized but interesting examples include two DHH proteins of Mycoplasma genitalium and Mycoplasma pneumoniae, located in operons encoding proteins required for adherence to the respiratory epithelium (32, 36, 64). Furthermore, SMU.1297, a DHH subfamily 1 protein of S. mutans, was recently found to be involved in superoxide stress tolerance by exposing wild-type and SMU.1297 mutant strains to menadione, a quinine compound that generates superoxide anions in bacteria (75). Analysis of the SMU.1297 sequence showed a high degree of homology to the B. subtilis YtqI protein, which possesses dual activities: oligoribonuclease (cleaves small RNAs smaller than 5-mers) and 3′-phosphoadenosine–5′-phosphate (pAp) phosphatase in vitro (46). Biochemical analysis of SMU.1297 demonstrated that it has pAp phosphatase but no oligoribonuclease activity (75). B. subtilis has another DHH/DHHA1 domain protein, YybT, which exhibits PDE activity toward cyclic dinucleotides (58). Based on their amino acid sequences, SP1298 is ∼68% identical to SMU.1297, while SP2205 shares 32% homology with YybT. SMU.1297 and YtqI consist of 310 and 313 amino acids, respectively, which is comparable to the size of SP1298, while the size of YybT (659 amino acids) is comparable to that of SP2205. Given these similarities, it is not unlikely that SP1298 and SP2205 may possess hydrolase and/or nuclease activity. Characterization of their biochemical function(s) will be a subject of future studies.

The combination of both DHH subfamily 1 antigens SP1298 and SP2205 provided the best protection (60%) against pneumococcal infection, while immunization with the individual DHH subfamily 1 antigens did not confer significant protection. For more than a decade, the protective potential of various pneumococcal virulence factors, such as Ply, PspA, PspC, and PsaA, either as single proteins or in combination, has been examined (1, 15, 40, 41, 49, 52, 71, 72). Two studies performed by Ogunniyi et al. have shown that in general, median survival times of mice immunized with different combinations of PspA, PsaA, PdB (a genetically detoxified derivative of Ply), and the pneumococcal histidine triad (Pht) protein PhtB or PhtE were significantly longer upon pneumococcal challenge than survival times of mice immunized with single antigens (49, 50). Briles et al. have shown that protection against pneumonia improved when mice were immunized with the combination of PspA and PdB, compared to results with single-protein immunization (15). These studies have convincingly demonstrated that a combination protein vaccine improves the level of protection against pneumococcal disease in experimental mouse models. Our study also supports the paradigm that a multicomponent protein vaccine will confer protection against invasive disease and perhaps against carriage as well. In our opinion, future experimental protein vaccine studies should focus on vaccine formulations that comprise protein antigens sharing complementary functions or even combinations of functional protein families in order to determine whether the protection will be magnified.

In conclusion, we have demonstrated that the two conserved pneumococcal DHH subfamily 1 proteins, SP1298 and SP2205, play a significant role in four important stages of pneumococcal pathogenesis, i.e., colonization, otitis media, pneumonia, and bacteremia, and that vaccination with a combination of the two proteins is able to confer protection against pneumococcal disease. While further research on these two proteins is needed to determine their cellular localization during pathogenesis and biochemical function and to evaluate the protective potential against other pneumococcal serotypes and genotypes, our data suggest that SP1298 and SP2205 are interesting candidates for future protein-based pneumococcal vaccines.

ACKNOWLEDGMENTS

We thank Sofie van Erk, Marilyn Bok, Christa E. van der Gaast-de Jongh, and Marc Eleveld for their technical assistance. We thank Jean-Pierre Claverys (CNRS-Université Paul Sabatier, Toulouse, France) for kindly providing the pCEP plasmid.

This work was financially supported by Pneumopath grant 222983 from the European Union Seventh Framework Program (FP7), the EC Sixth Framework Program, and Horizon Breakthrough grant 93518023 from the Netherlands Genomics Initiative.

FOOTNOTES

    • Received 29 December 2010.
    • Returned for modification 22 February 2011.
    • Accepted 5 July 2011.
    • Accepted manuscript posted online 18 July 2011.

  • † Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01383-10.

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