ABSTRACT
Streptococcus pneumoniae is a commensal of the human nasopharynx but can cause invasive diseases, including otitis media, pneumonia, sepsis, and meningitis. The capsular polysaccharide (capsule) is a critical virulence factor required for both asymptomatic colonization and invasive disease, yet the expression level is different in each anatomical site. During colonization, reduced levels of capsule promote binding to the host epithelium and biofilm formation, while during systemic infection, increased capsule is required to evade opsonophagocytosis. How this regulation of capsule expression occurs is incompletely understood. To investigate the contribution of transcriptional regulation on capsule level in the serotype 4 strain TIGR4, we constructed two mutants harboring a constitutive promoter that was either comparably weaker (Pcat) or stronger (PtRNAGlu) than the wild-type (WT) capsule promoter, Pcps. Mild reductions in cpsA and cpsE transcript levels in the Pcat promoter mutant resulted in a 2-fold reduction in total amounts of capsule and in avirulence in murine models of lung and blood infection. Additionally, the PtRNAGlu mutant revealed that, despite expressing enhanced levels of cpsA and cpsE and possessing levels of capsule comparable to those of WT TIGR4, it was still significantly attenuated in all tested in vivo niches. Further analysis using chimeric promoter mutants revealed that the WT −10 and −35 boxes are required for optimal nasopharyngeal colonization and virulence. These data support the hypothesis that dynamic transcriptional regulation of the capsule operon is required and that the core promoter region plays a central role in fine-tuning levels of capsule to promote colonization and invasive disease.
INTRODUCTION
Streptococcus pneumoniae (pneumococcus) is a Gram-positive bacterium that asymptomatically colonizes human nasal passages. However, it can cause potentially devastating diseases such as pneumonia, sepsis, and meningitis if it gains access to the lungs, blood, or central nervous system (1–3). As a consequence of these pneumococcal diseases, approximately 1 million children, primarily in developing parts of the world, die each year (2, 4). In addition, a similar number of adults, particularly the elderly, die each year from invasive pneumococcal diseases (5). The capsular polysaccharide (capsule) is a critical virulence factor required for both asymptomatic colonization and invasive disease (1, 6, 7). Interestingly, the amount of capsule present on the surface of S. pneumoniae in each anatomical niche is strikingly different (8–11). Capsule facilitates pneumococcal passage through the mucus layer in the nasopharynx and prevents detection by host immune cells (1, 12). Once S. pneumoniae passes through the mucus layer, it likely expresses less capsule in order to expose underlying surface molecules that promote binding to epithelial cells and the formation of bacterial aggregates called biofilms (9, 13, 14). Biofilms further protect S. pneumoniae from immune surveillance and antibiotic treatment and allow long-term persistence in this niche (15, 16). However, during systemic blood infection, S. pneumoniae must increase capsule expression in order to evade destruction mediated by complement-mediated opsonophagocytosis (17–19). Survival in these different niches thus requires tight control of capsule expression levels. However, the exact molecular mechanisms responsible for regulating capsule expression are unknown.
Earlier work demonstrated that the cps locus is likely transcribed as an operon from a single promoter upstream of cpsA (20) and that in the majority of the >90 capsule serotypes, cpsA-cpsD (cpsA-D) are highly conserved and contribute to modulating capsule levels (7, 21, 22). Genes downstream of cpsD encode enzymes required for serotype-specific synthesis, polymerization, and export of capsular polysaccharide (7, 23, 24) (Fig. 1A). One potential mechanism by which S. pneumoniae regulates the amount of capsule is at the posttranslational level via a multiprotein phosphorelay system encoded by cpsA-D (7, 24). Although the role of CpsA is largely unknown, previous work demonstrated that CpsC and CpsD are the transmembrane and cytoplasmic domains of an autophosphorylating protein tyrosine kinase, respectively, while CpsB serves as a phosphotyrosine phosphatase that controls the phosphorylation status of CpsD (25, 26). While it has been shown that an intact phosphorelay system is needed for production of wild-type amounts of full-length capsule (27), it remains unclear if the phosphorylation state of CpsD positively or negatively affects production of capsule (28–30).
Genetic architecture of the cps locus in S. pneumoniae and schematic of DNA constructs used to generate cps promoter mutant strains and promoter sequences. (A) The locus shown is representative of the Wzx-Wzy-dependent capsule cassette of serotype 4. The capsule locus, ∼15 kb in size, is located between the conserved genes dexB and aliA and is flanked on either side by insertion sequences (IS). cpsA-D are highly conserved across most serotypes. Serotype-specific genes encode all enzymes required to synthesize serotype-specific capsular polysaccharides. (B) Using the depicted DNA constructs, the WT TIGR4 capsule promoter (solid line) was replaced with the mutant promoter PtRNAGlu (dotted line, strong constitutive; Spcr) or Pcat (weak constitutive; Cmr) by allelic exchange. Chimeric promoter mutants PcpsCore and PtRNAGluCore (both Spcr) were generated in the same fashion. For the pseudorevertant PtRNAGluRev (Cmr) and PcatRev (Spcr) strains, the respective mutant strain was transformed with a DNA construct containing the native capsule promoter. The stem-loop represents a bidirectional terminator. (C) Sequences of the promoters for WT Pcps, PtRNAGlu (strong constitutive), Pcat (weak constitutive), and chimeric mutants PcpsCore and PtRNAGluCore. The transcriptional start site for the WT promoter Pcps was experimentally determined using 5′ RACE and is indicated by the solid-line arrowhead. Putative transcriptional start sites in Pcat, PtRNAGlu, and PtRNAGluCore promoter mutants are indicated by the dashed-line arrowheads. Nucleotide differences relative to Pcps are bolded.
S. pneumoniae may also alter levels of capsule via phase variation between two distinct phenotypes: opaque and transparent. The opaque phenotype is characterized by increased amounts of capsule, which promotes virulence in the blood due to enhanced resistance to opsonophagocytosis. Conversely, the transparent variant possesses less capsule and more exposed cell wall phosphorylcholine (P-Cho) and surface proteins and is consequently better at binding epithelial cells and colonizing the nasopharynx (10, 11, 17, 18). While the mechanism(s) responsible for capsule phase variation remains unclear (31–33), it is likely that this process provides S. pneumoniae with a way to rapidly alter capsule level in order to adapt to dynamic environments and immune pressures experienced during infection.
One potential means of controlling the amount of capsule is at the transcriptional level. Previous work revealed that levels of capsule transcripts were significantly enhanced in a mouse model of blood infection, supporting the notion that S. pneumoniae evades opsonophagocytic killing by increasing capsule (34). In order to examine whether the native TIGR4 capsule promoter, Pcps, was required for fine-tuning capsule expression level, we replaced it with a comparably weaker (Pcat) or stronger (PtRNAGlu) constitutively active promoter derived from the chloramphenicol resistance gene from pAC1000 or S. pneumoniae TIGR4 tRNA-Glu-1 gene, respectively. Expression of capsule transcripts, total amounts of capsular polysaccharide, resistance to C3 complement deposition, and exposed P-Cho were determined for each of the cps promoter mutants. Competition experiments between TIGR4 and Pcat or PtRNAGlu mutants in murine models of colonization and invasive disease in the lungs and blood revealed that dynamic control of the capsule operon via transcriptional control was required for optimal colonization and virulence. Additional experiments using two chimeric promoter mutants, PcpsCore, which contains the Pcps core promoter sequence and upstream sequence from the PtRNAGlu promoter, and PtRNAGluCore, which contains the PtRNAGlu core promoter sequence and upstream sequence from Pcps, demonstrated that the wild-type −10 and −35 hexamers are key to colonization and virulence.
MATERIALS AND METHODS
Bacterial strains and growth conditions.S. pneumoniae TIGR4 (serotype 4) and its unencapsulated derivative AC4421 were from our laboratory stock. For the experiments indicated in the text, D39 (serotype 2) was also used.
S. pneumoniae was grown in Todd-Hewitt yeast extract (THY) medium at 37°C in a 5% CO2 incubator. THY medium was Todd-Hewitt broth (Becton, Dickinson, Co.) supplemented with 0.5% yeast extract (Fischer Scientific, Inc.) and 5 μl/ml Oxyrase (Oxyrase, Inc.). Growth on plates was done on blood agar (BA) plates at 37°C in a 5% CO2 incubator. BA plates were tryptic soy agar plates supplemented with 5% (vol/vol) sheep blood (Northeast Laboratories, Inc.). Where appropriate during growth in THY medium or on BA plates, antibiotics at the following concentrations were included: spectinomycin (Spc) at 200 μg/ml and chloramphenicol (Cm) at 4 μg/ml.
5′ RACE to identify the transcriptional start site of the capsule promoter.To identify the transcriptional start site of Pcps shown in Fig. 1C, a First Choice RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) kit (Ambion, Inc.) was used per the manufacturer's instructions. Briefly, DNase-treated RNA was prepared from mid-exponential-growth-phase TIGR4 cells grown in THY medium (optical density at 600 nm [OD600] of 0.6 to 0.8) and subsequently treated with calf intestine phosphatase. After phenol-chloroform extraction, RNA was incubated with tobacco acid pyrophosphatase to yield a free 5′ monophosphate used for adapter ligation using T4 RNA ligase. Samples were reverse transcribed and used as a template for nested PCR with forward adapter and reverse cpsA specific primers (Table 1). The resultant amplicon was sequenced by the Tufts University Core Facility (TUCF) to determine the transcription start site.
Relevant plasmids and primers used in this study
Generation of promoter mutant strains.Promoter mutant strains illustrated in Fig. 1B were generated by allelic exchange. Each allelic exchange construct was generated in vitro using splicing by overlap extension PCR (35). The upstream and downstream arms of homology flanking the new sequences were PCR amplified from TIGR4 genomic DNA (gDNA). To aid in making these promoter replacements, Spcr and Cmr cassettes were incorporated into the constructs to allow direct selection of transformants. These antibiotic resistance cassettes were PCR amplified using plasmids pAC1294 and pAC100, respectively (Table 1), as templates (36–38). In each mutant construction, the three insertion (IS) elements upstream of the capsule promoter were deleted to avoid recombination of the replacement construct with identical IS elements scattered throughout the genome. The cpsA promoter in D39 was replaced with Pcat using the same strategy. Transformation of S. pneumoniae was done as previously described (39). All promoter replacement mutations were confirmed by DNA sequencing.
RNA isolation and cDNA synthesis.Total RNA was isolated from 1 ml of mid-exponential-growth-phase bacteria. Cell pellets were snap frozen in liquid nitrogen and stored at −80°C until use. Pellets were resuspended in 1 ml of TRIzol (Invitrogen, Corp.) with 4 μl/ml of glycogen (5 mg/ml; Ambion, Inc.) and added to O-ring tubes with 400 μl of 0.1-mm Zirconia beads (Bio Spec, Inc.). Cells were lysed using a bead beater, and RNA was extracted per the TRIzol manufacturer's instructions, followed by ethanol precipitation. Samples were further purified using a Qiagen RNeasy Kit (Qiagen, Inc.), eluted in 32 μl of diethyl pyrocarbonate (DEPC)-treated water (Ambion, Inc.), and DNase treated using a Turbo DNase-free kit (Ambion, Inc.) for 1 h at 37°C. One microgram of the resulting RNA was reverse transcribed in a 20-μl reaction mixture using an iScript cDNA synthesis kit (Bio-Rad, Inc.) with the following parameters: 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min. In parallel, for each sample, controls lacking reverse transcriptase (RT) were run to ensure that subsequent quantitative reverse transcription-PCR (qRT-PCR) analysis was free of contaminating gDNA. Subsequently, samples were diluted 2-fold with DEPC-treated water and stored at −20°C until analysis.
Quantitation of capsule gene transcription.To determine relative expression levels of genes in the capsule operon, quantitative reverse transcription-PCR (qRT-PCR) was done using primers specific for cpsA and cpsE and the housekeeping gene rplI (a constitutively expressed ribosomal protein) (Table 1) that were designed using Primer 3 software (40). SYBR green Super Mix (2×; Bio-Rad, Inc.) reaction mixtures contained 1 μl each of forward and reverse primers (10 μM) and 1 μl of cDNA template. Using the housekeeping gene rplI as an internal control, relative expression levels were calculated using the average mean cycle threshold (CT) value for rplI and the gene of interest for each sample and the equation 1.8e(CT rplI − CT gene of interest) (41).
Immunodot blot assays.For the purpose of quantifying the amount of capsule present in wild-type and promoter mutant strains using immunodot blotting, 1 ml of OD600-matched mid-exponential-growth-phase bacteria was pelleted and stored at −20°C until use. Samples were resuspended in 300 μl of cell wall lysis buffer (50 mM Tris, pH 7.5, 1 mg/ml lysozyme, 300 U/ml mutanolysin [Sigma-Aldrich, Co.]) and incubated at 37°C for 30 min. Subsequently, samples were sonicated for 3 min at 4°C at maximum amplitude using a water bath sonicator (Branson, Inc.). Samples were 2-fold serially diluted in phosphate-buffered saline (PBS), and 5 μl was spotted on 0.2-μm-pore-size nitrocellulose membranes (Invitrogen, Inc.) with suction. Membranes were processed according to the SNAP i.d. Protein Detection System (Millipore, Corp.) instructions using rabbit anti-serotype 4 serum (Statens Serum Institut) and Cy5-conjugated goat anti-rabbit secondary antibody (Invitrogen, Inc.), both at a dilution of 1:600. Membranes were scanned on a Fuji scanner, and relative fluorescence as normalized to a blank was calculated using MultiGauge analysis software (Fujifilm, Corp.). To ensure that differences in capsule intensity were not due to differences in total amounts of cells among samples, aliquots were subjected to protein analysis using a MicroBCA Protein Kit (Thermo Scientific, Inc.).
C3 deposition, exposed phosphocholine (P-Cho) assays, and fluorescence-activated cell sorter (FACS) analysis.For C3 deposition assays, 1 ml of mid-exponential-growth-phase bacteria was pelleted, washed in PBS, and resuspended in 500 μl of Hanks buffer with Ca2+ and Mg2+ (Gibco, Corp.) supplemented with 0.1% gelatin (Fischer Scientific, Inc.). C3 deposition reaction mixtures (150 μl) were comprised of 107 CFU in 50 μl and 100 μl of infant rabbit serum to a final concentration of 10% (AbD Serotec, Co.). Samples were incubated in a 37°C rolling incubator for 30 min. Next, opsonization reaction mixtures were chilled for 3 min on ice, quenched with 500 μl of Hanks buffer without Ca2+ and Mg2+ (Gibco) with 0.1% gelatin and pelleted at 4,000 rpm for 5 min. Pellets were resuspended in fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit C3 antibody in 100 μl of Hanks buffer without Ca2+ and Mg2+ with 0.1% gelatin at 1:200 (MP Biomedicals) and incubated on ice in the dark for 30 min. Staining reactions were quenched with 500 μl of Hanks buffer without Ca2+ and Mg2+ with 0.1% gelatin and centrifuged at 4,000 rpm for 5 min, and pellets were resuspended in 300 μl of 2% paraformaldehyde (PFA; Sigma-Aldrich, Co.). Samples were collected (25,000 events) on a FACSCalibur analytical flow cytometer.
For exposed P-Cho assays, 300 μl of mid-exponential-growth-phase bacteria was pelleted and washed in PBS. Pellets were resuspended in 100 μl of unconjugated mouse IgA anti-P-Cho at 1:100 in 1× PBS and incubated on ice for 30 min. Samples were quenched with 500 μl of 1× PBS and centrifuged at 4,000 rpm for 5 min. Pellets were resuspended in 100 μl of phycoerythrin (PE)-conjugated rat anti-mouse IgA secondary antibody at 1:100 in PBS and kept on ice, in the dark, for 30 min. Staining reactions were quenched with 500 μl PBS, and products were pelleted, resuspended in 300 μl of 2% PFA and analyzed as described above. All FACS data were analyzed and plotted using Flowlogic (Inivai Technologies).
Animal infections.All procedures involving mice were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts University School of Medicine. All competition experiments used female Swiss Webster mice, 6 to 9 weeks old (Taconic Laboratories). TIGR4 or D39 and isogenic promoter mutant strains of interest were grown to mid-exponential phase in THY medium and mixed at a 1:1 ratio. For nasopharyngeal colonization and lung infection, isoflurane-anesthetized mice were inoculated with 10 μl (5 μl/nare; 109 CFU/ml) and 40 μl (2.5 × 108 CFU/ml) of the 1:1 mix, respectively. For blood infections, mice received ∼104 CFU in 100 μl by intraperitoneal (i.p.) injection. Mice were euthanized at the appropriate time points (5 days after nasopharyngeal colonization, 36 to 48 h after lung infection, and 24 to 36 h after blood infection) by CO2 asphyxiation. Nasal flushes were performed with 500 μl of sterile PBS. Lungs were perfused with 10 ml of PBS, aseptically removed, and homogenized in 1 ml of sterile PBS. To recover bacteria from the blood, 500 μl of blood was removed by cardiac puncture, and clotting was prevented with 3 μl of 500 mM EDTA. Serial dilutions of recovered bacteria from each mouse were plated on BA and BA supplemented with Spc or Cm where appropriate. The competitive index (CI) of each promoter mutant strain was calculated using the following equation: (mutant output CFU/WT output CFU)/(mutant input CFU/WT input CFU). On the occasion that no mutant bacteria were recovered from an animal, the numerator was given a value of 1 in order to calculate a maximum CI value.
To rule out the possibility that any observed in vivo attenuation in promoter mutant strains was due to a defect in overall viability, in vitro competition experiments were performed between wild-type TIGR4 and Pcat, PtRNAGlu, or PtRNAGluCore strains. Wild-type and promoter mutant strains were grown to mid-exponential phase in THY medium; 1-ml aliquots were pelleted and resuspended in THY medium. Strains were mixed at a 1:1 ratio, serially diluted, and differentially plated on BA and BA plus the appropriate antibiotic (Spc or Cm) to determine the input ratio of mutant/wild type. Subsequently, the 1:1 mix was inoculated at a 1:100 dilution into THY medium and grown to mid-exponential phase (∼4 h). A volume of bacteria was serially diluted and differentially plated on BA and BA plus antibiotic to calculate the output ratio of wild-type versus promoter mutant bacteria at the end of the competition. The CI was determined by the same equation described for the in vivo experiments above. All promoter mutants demonstrated no in vitro growth defect compared to the wild type (data not shown).
Statistical analysis.Wilcox signed-rank tests and one-way analysis of variance (ANOVA) statistical tests were performed where indicated in the figure legends using GraphPad Prism (GraphPad Software, Inc.).
RESULTS
Generating cps promoter mutant strains and evaluating in vitro levels of capsule gene expression. S. pneumoniae occupies drastically different anatomical niches during nasopharyngeal colonization and invasive disease in the blood or lungs (8–11). The amounts of capsule present on the pneumococcal surface in each site have been reported to be markedly different. This supports the hypothesis that S. pneumoniae tightly controls the level of capsule expression in order to optimize fitness. One potential means by which S. pneumoniae controls the amount of capsule is at the transcriptional level (34). A bioinformatics approach was previously taken to identify the promoter elements in the sequence immediately upstream of the first gene in the capsule operon, cpsA (Fig. 1A) (42). However, there has been no experimental data reported that identify the transcriptional start site and promoter sequences for the capsular operon. To do so, we performed 5′ RACE on RNA isolated from mid-exponential-growth-phase TIGR4 bacteria. As shown in Fig. 1C, data from this experiment located the transcription start site to a position 12 bp downstream from the start of a consensus −10 box and 35 bp downstream of a near consensus sigma-70 Pribnow box or −35 box which contained one mismatch from the consensus, TTGACA. Of note, the 17-bp spacing between the −10 and −35 boxes differs from the optimal 16-bp spacing (43, 44). We designated these the core elements of the native Pcps in TIGR4, and we utilized this information to construct a panel of cps promoter replacements to determine how transcriptional regulation impacts capsule expression as well as colonization and virulence phenotypes. Two promoter replacement mutants, Pcat and PtRNAGlu, were constructed by replacing the entire intergenic region and divergent IS elements upstream of cpsA. We showed that Pcat and PtRNAGlu constituted weak and strong constitutively active promoters relative to Pcps, respectively (Fig. 2). A priori, Pcat was predicted to be weaker than Pcps due to its −35 box having two mismatches from the consensus, while PtRNAGlu was predicted to be a stronger promoter based on its consensus −10/−35 boxes, ideal spacing of 16 bp, and presence of an extended −10 sequence (TG motif) known to stabilize RNA polymerase-DNA interactions (43–46). Additionally, we constructed two chimeric promoter mutant strains, the PcpsCore mutant, which possesses the wild-type Pcps core promoter (−35 to start site) paired with the PtRNAGlu sequence upstream of the −35 box, and the PtRNAGluCore mutant, which possesses the wild-type sequence from downstream of the deleted IS elements to the −35 box paired with the core promoter including the extended −35 box from PtRNAGlu (Fig. 1B and C). This panel of mutants allowed us to investigate the following: (i) how modulation of capsule transcripts impacts amounts of capsular polysaccharide on the cell surface, (ii) if dynamic control of capsule genes is required for optimal colonization and invasive disease, and (iii) which region(s) of the native Pcps promoter is critical for colonization and virulence.
Capsule gene expression in cps promoter mutant strains. Promoter mutant strains were grown to mid-exponential phase in THY. Total RNA was extracted from 1-ml aliquots and reverse transcribed into cDNA. qRT-PCR was performed using primers specific for cpsA or cpsE, and results were normalized to the housekeeping gene rplI. Data are presented as relative units compared to the WT (TIGR4 or D39). Data shown for TIGR4, Pcat, and PtRNAGlu are means ± standard deviations of three to five independent experiments. ***, P < 0.001, compared to TIGR4 using one-way ANOVA. Data shown for PcatRev, PtRNAGluRev, D39, D39 Pcat mutant, and chimeric promoter mutants are means ± standard deviations of two to three independent experiments.
To evaluate the effect of the mutant cps promoters on capsule transcript levels, we performed qRT-PCR analysis on mid-exponential-growth-phase bacteria using primers for cpsA and cpsE (Fig. 2). Gene expression was normalized to rplI, and values shown are relative units compared to TIGR4. As anticipated, the mutant strain containing a strong constitutively active promoter, PtRNAGlu, expressed approximately 2-fold higher levels of cpsA and cpsE than TIGR4 (Fig. 2). Importantly, the pseudorevertant PtRNAGluRev strain, in which the native promoter and upstream sequences were restored while the antibiotic cassette was retained (Fig. 1B), expressed WT levels of capsule transcripts, thus confirming that any observed phenotype was due to the promoter replacement and not to the presence of the cis-encoded antibiotic cassette, deletion of the IS elements, or to second site mutations. Although not statistically significant, the mutant strain containing a weak constitutively active promoter, Pcat, exhibited slightly reduced levels of cpsA and cpsE, which was also observed in a serotype 2 strain, D39, suggesting that this phenotype is not serotype specific (Fig. 2). The PcpsCore chimeric promoter mutant expressed levels of cpsA comparable to the TIGR4 level. Surprisingly, the PtRNAGluCore mutant had a lower expression level of cpsA than its parental PtRNAGlu strain. It is possible that a synergistic interaction between the core promoter sequence and the upstream element is required for increased expression, and its disruption in the PtRNAGluCore strain results in an expression level similar to that of wild type. Taken together, these findings indicate that the presence of a promoter with various strengths upstream of the capsule operon can accordingly influence expression levels of cps transcripts.
Characterization of capsular polysaccharide on cps promoter mutant strains using C3 deposition, exposed phosphocholine, and immunodot blot assays.Previous work demonstrated that S. pneumoniae serotypes possessing enhanced levels of capsule were more resistant to complement C3 deposition and subsequent phagocytosis by neutrophils, while less encapsulated strains were extremely sensitive (17, 47–49). Additionally, resistance to complement deposition was shown to correlate with enhanced virulence in an animal model of otitis media (50), supporting the notion that the capsule is a critical virulence factor that facilitates the development of disease. With this knowledge, we used a FACS-based complement C3 deposition assay to indirectly quantify the amounts of capsule on cps promoter mutant strains. For these experiments, mid-exponential-growth-phase bacteria were subjected to in vitro opsonization assays with infant rabbit serum (a rich source of complement C3) and subsequent staining with anti-C3 antibody followed by flow cytometric analysis. As expected, an unencapsulated mutant strain of S. pneumoniae, the Δcps strain, was the most sensitive to C3 deposition and exhibited ∼60-fold larger amounts of C3 deposition than TIGR4 (Fig. 3A and D). In accordance with the qRT-PCR data that revealed a mild reduction in cpsA and cpsE gene expression, the Pcat promoter mutant was approximately 3-fold more susceptible to C3 deposition, indicating that it possesses reduced capsule expression (Fig. 3A, B, and D), while the PcatRev strain recovers the wild-type C3 deposition phenotype (Fig. 3B and D). Despite the significant increase in capsule gene transcript levels, the PtRNAGlu promoter mutant strain did not exhibit enhanced resistance to C3 deposition compared to WT TIGR4 as we might have anticipated (Fig. 3A and D). Additionally the PcpsCore and PtRNAGluCore chimeric promoter mutant strains demonstrated amounts of C3 deposition comparable to the amount of TIGR4 (Fig. 3 C and D).
Complement C3 deposition on cps promoter mutant strains of S. pneumoniae. Mid-exponential-growth-phase bacteria were incubated with 10% infant rabbit serum, and C3 deposition on pneumococcal cells was determined by flow cytometry using an FITC-conjugated goat anti-rabbit C3 antibody. (A) Representative histogram overlay plots comparing the C3 deposition phenotypes of WT TIGR4, Δcps, Pcat, and PtRNAGlu promoter mutant strains. (B and C) Representative histogram overlay plots, from a different independent experiment, comparing C3 deposition phenotypes of WT TIGR4, Pcat, and PcatRev or of WT TIGR4, PcpsCore, and PtRNAGluCore. (D) Mean fluorescence intensity (MFI) of the C3+ population, shown as fold increase relative to WT TIGR4 ± standard deviations from three to five independent experiments. *, P < 0.05, compared to TIGR4 using one-way ANOVA.
In another approach to assess capsule-related phenotypes of cps promoter mutant strains, we examined the amount of exposed P-Cho on mid-exponential-growth-phase bacteria using flow cytometry. Akin to the inverse relationship between amount of capsule and C3 deposition, opaque variants of S. pneumoniae have thicker capsules and less exposed P-Cho, while transparent variants have less capsule and consequently possess more exposed P-Cho (10, 17, 18). Our experiments corroborated the results from the C3 deposition assays and revealed that both the Δcps and Pcat promoter mutant strains have significantly higher levels of exposed P-Cho than TIGR4, while the PcatRev strain was restored to the TIGR4 level (Fig. 4A and B). Again, despite the augmented levels of cps transcripts in the PtRNAGlu strain, this did not result in reduced levels of exposed P-Cho (Fig. 4A and B). Additionally, the PcpsCore and PtRNAGluCore chimeric promoter mutant strains exhibited P-Cho phenotypes similar to the phenotype of TIGR4 (Fig. 4B).
Exposed phosphocholine on cps promoter mutant strains. WT TIGR4 and cps promoter mutant strains were grown to mid-exponential phase and stained with an unconjugated TEPC15 mouse IgA anti-phosphocholine antibody, followed by a PE-conjugated rat anti-mouse secondary antibody. Samples were fixed in 2% PFA and read on a FACSCalibur flow cytometer. (A) A representative histogram plot of the mean fluorescence intensity of TEPC15 antibody binding to exposed phosphocholine (P-Cho+) on WT TIGR4, Δcps, Pcat, and PtRNAGlu promoter mutant strains. The gate was set based on a negative control that was treated with secondary antibody only. (B) Mean fluorescence intensity (MFI) of exposed P-Cho bound by TEPC15 antibody on WT TIGR4, Δcps, and capsule promoter mutant strains. Data shown for TIGR4, Δcps, Pcat, PtRNAGlu, and PcatRev strains are means ± standard deviations of three to four independent experiments; data for chimeric promoter mutants are from two independent experiments. ***, P < 0.001, compared to TIGR4 using one-way ANOVA.
In a third assay we directly quantified the amount of capsule present on the cps promoter mutant strains using an immunodot blot assay. Bacteria were grown to mid-exponential phase in THY medium, adjusted to identical optical densities, and subsequently 2-fold serially diluted and transferred to a nitrocellulose membrane. In additional experiments, samples were normalized to cell number or to total protein content to ensure that any differences in capsule signal were not due to discrepancies between optical density and these other valid measurements (data not shown). Blots were developed using unconjugated rabbit anti-serotype 4 serum and a Cy5-conjugated goat anti-rabbit secondary antibody. The Pcat promoter mutant strain possessed approximately 2-fold less capsule than TIGR4, while the PtRNAGlu strain exhibited comparable levels of capsular polysaccharide (Fig. 5A and B). The pseudorevertant PcatRev strain restored capsule levels back to the wild-type level. Taken together, these data reveal that even a mild reduction in cps transcripts, observed in the Pcat strain, can result in a significant phenotype related to the amount of capsule found on the cell surface. Conversely, overexpression of capsule genes in the PtRNAGlu strain may not directly result in overproduction of capsule. It may be possible that other regulatory mechanisms such as the phosphorelay system encoded by cpsA-D contribute to the total amount of capsular polysaccharide on the cell surface (24, 25, 28, 29).
Quantitative immunodot blot analysis of total capsule in cps promoter mutant strains. Strains of interest were grown to mid-exponential phase in THY medium; 1-ml aliquots were pelleted and resuspended in 1× PBS to match OD600 values across samples. Samples were serially 2-fold diluted, and 5 μl was spotted onto a nitrocellulose membrane with suction. Membranes were developed using rabbit anti-serotype 4 sera and a Cy5-conjugated goat anti-rabbit secondary antibody. Blots were read, and relative fluorescence units were calculated using a Fuji Imager and MultiGauge software. (A) Representative immunodot blots from two independent experiments showing a 2-fold dilution series for each strain. (B) Quantitation of relative fluorescence intensity compared to a blank lane relative to the WT TIGR4. Data shown for TIGR4, Δcps, Pcat, and PtRNAGlu strains are means ± standard deviations from four independent experiments; data for the PcatRev strain are from two independent experiments. **, P < 0.01, and ***, P < 0.001, compared to TIGR4 using one-way ANOVA.
Analysis of cps promoter mutant strains in murine models of nasopharyngeal colonization and invasive disease.Based on our hypothesis that S. pneumoniae exerts transcriptional control of the capsule expression level in order to thrive in different host niches, we tested the panel of cps promoter mutants in murine models of colonization and invasive disease in the lung and blood. For these experiments, we competed TIGR4 against each of the cps promoter mutant strains. These experiments revealed that despite only a 2-fold reduction in capsule (Fig. 5A and B), the Pcat promoter mutant strain was severely attenuated in all tested niches (Fig. 6A). The Pcat strain was attenuated 50-fold in the nasopharynx, while in the lungs and blood we failed to recover any of the mutant, indicating that it was completely avirulent in these niches (Fig. 6A). To ensure that the inability to recover Pcat bacteria from the blood following i.p. inoculation was not due to a defect in the bacteria's ability to escape the peritoneal cavity and gain access to the blood, we repeated these experiments using intravenous (i.v.) injections. Regardless of route of administration, there were no recovered Pcat mutant cells in the bacteremia model of infection (data not shown). Importantly, the PcatRev pseudorevertant strain was restored to full virulence in all three animal infection models (Fig. 6A). Given the striking loss of virulence of the Pcat mutant in the TIGR4 strain background, we tested whether the same cps promoter mutation in another capsular serotype strain would yield the same phenotype. For this we evaluated the Pcat mutant promoter in the serotype 2 strain, D39. Similar to data obtained in the TIGR4 background, the D39 Pcat mutant demonstrated an 8-fold defect in the nasopharynx and complete attenuation in the blood compared to the WT D39 (Fig. 6B).
PtRNAGlu and Pcat promoter mutant strains are attenuated during in vivo colonization and invasive disease. Competition experiments between the WT TIGR4 and Pcat (A) or PtRNAGlu (C) strain were conducted in the nasopharynx (Naso), lung, and blood. (B) D39 Pcat mutant versus WT D39 competition experiments were performed in the nasopharynx and blood. In all experiments, mutant and WT bacteria were grown to mid-exponential phase, washed in 1× PBS, mixed 1:1, and inoculated into Swiss Webster mice (107 CFU/mouse, nasopharynx and lungs; 104 CFU/mouse, blood). Mice were euthanized, and bacterial counts in nasopharyngeal lavage fluids, lung homogenates, or blood were determined by plating for CFU. CI values were calculated by dividing the numbers of CFU for the cps promoter mutant by those for the WT. The horizontal bar shows the median for each group. Each point represents an individual mouse. Open symbols indicate a mouse that yielded no detectable mutant colonies; a value of 1 was assigned for the numerator in order to determine a maximum CI value for these animals. **, P < 0.01, using Wilcox signed-rank tests.
Remarkably, despite exhibiting a comparable level of capsule expression to TIGR4 in vitro, the constitutive PtRNAGlu promoter mutant demonstrated ∼10-fold attenuation in models of nasopharyngeal colonization and invasive disease in the lungs and blood (Fig. 6C). The pseudorevertant PtRNAGluRev strain was restored for colonization and virulence. These data indicate that, despite the wild-type amount of capsule provided by the PtRNAGlu promoter during growth in vitro, the levels of expression in vivo are not optimal for colonization and systemic infection. Although several possible explanations for these observations exist, we favor the possibility that this promoter lacks the dynamic transcriptional regulation afforded by the native Pcps promoter in vivo needed to ensure that optimal amounts of capsule are made in each host niche.
Examining regions of Pcps necessary for colonization and virulence.Based on our observations from the in vivo competition experiments between the TIGR4 strain and cps Pcat and PtRNAGlu promoter mutants, we hypothesized that some characteristic afforded by the Pcps promoter, such as dynamic transcriptional regulation of cps genes or possibly stochasticity of the promoter itself, is required for optimal colonization and virulence. To start examining regions of the Pcps promoter involved in controlling capsule expression to allow for the aforementioned in vivo processes, we generated two chimeric promoter mutant strains, the PcpsCore and PtRNAGluCore strains, which are designed to test the role of the Pcps core elements. The PcpsCore promoter contains native (Pcps) sequence starting from the −35 hexamer and proceeding downstream, paired with the upstream sequence from the PtRNAGlu promoter, while the PtRNAGluCore strain harbors PtRNAGlu sequence starting from the −35 hexamer and proceeding downstream, paired with the native upstream sequence (Fig. 1B and C). For these experiments, in vivo competitions between TIGR4 and the PcpsCore or PtRNAGluCore strain were performed in the nasopharynx and blood. As shown in Fig. 7A and B, the PcpsCore chimeric promoter mutant exhibited wild-type levels of both colonization and virulence. However, the PtRNAGluCore mutant was attenuated to the same extent as the PtRNAGlu mutant in both tested niches (Fig. 6C and 7). These data indicate that the wild-type −10/−35 promoter elements play a central role in regulating transcription of the cps operon to allow for optimal colonization and invasive disease.
Core promoter of Pcps is necessary and sufficient for intact colonization and virulence. In vivo competition experiments between WT TIGR4 and the chimeric PcpsCore or PtRNAGluCore promoter mutant were performed in the nasopharynx and blood. For all experiments, WT TIGR4 and PcpsCore or PtRNAGluCore mutant strains were grown to mid-exponential phase in THY medium, washed in 1× PBS, and mixed at a 1:1 ratio. Swiss Webster mice were inoculated either intranasally (107 CFU/mouse) or i.p. (104 CFU/mouse) and euthanized at the appropriate time point, and bacteria were recovered from nasopharyngeal lavage fluids or blood for enumeration. CI values were calculated by dividing the numbers of CFU for the chimeric promoter mutant by those for TIGR4. The horizontal bar shows the median for each group. Each point represents an individual mouse. **, P < 0.01, using Wilcox signed-rank tests.
DISCUSSION
The capsular polysaccharide is a critical virulence factor required for effective S. pneumoniae colonization of the nasal passages and invasive disease in the lungs and blood (1, 6, 7). Previous work highlights the need to fine-tune levels of capsule expression during these different stages within the host. During nasopharyngeal colonization, an initial amount of capsule is necessary to prevent mucus-mediated clearance (12), but this transitions to a scenario where less capsule is favored to expose underlying surface molecules that facilitate adherence to epithelial cells and promote biofilm formation (9, 13, 14, 51). Conversely, during invasive disease, enhanced levels of capsule are necessary to resist C3-mediated opsonophagocytic killing (11, 17, 18, 47). Taken together, these data demonstrate that S. pneumoniae must effectively fluctuate between two strikingly different capsule phenotypes, yet the underlying regulatory mechanisms are poorly understood.
In this study, we examined the role of transcriptional regulation as a potential mechanism of controlling capsule expression. To do so, we constructed two cps promoter mutants, each of which harbored a constitutively active promoter driving transcription of the capsule operon. As expected, the PtRNAGlu mutant strain, with a strong constitutively active promoter compared to the native promoter Pcps, exhibited augmented cps transcripts, while the weaker mutant promoter Pcat yielded a mild reduction in capsule transcripts. Although the PtRNAGlu mutant demonstrated elevated cps transcripts, in vitro characterization experiments using C3 deposition, exposed P-Cho, and immunodot blot assays revealed that this failed to result in more total capsular polysaccharide on the cell. It is possible that despite the presence of more cps transcripts, the cell may possess a maximum synthesis or export limit that has already been achieved, and, consequently, the presence of more transcripts fails to yield more capsule on the cell surface. Surprisingly, despite only a modest reduction in cpsA and cpsE expression levels, the Pcat mutant exhibited a major (2-fold) reduction in capsule expression. These data suggest that posttranscriptional regulatory mechanisms, such as those mediated by the ill-defined CpsA-D phosphorelay system, may also play a role in controlling capsule levels that could either prevent the increase of capsular polysaccharide in the presence of more transcripts or accentuate mild decreases in cps transcripts (7, 28, 29). Additionally, certain environmental conditions such as the availability of oxygen may also play a role in posttranscriptional regulation of capsule (30).
In regard to the effect of capsule expression on colonization and invasive disease, our data suggest that modulation of capsular polysaccharide, with either an increase or decrease in capsule levels, is critical. It was somewhat expected that a 2-fold reduction in capsular polysaccharide in the Pcat promoter mutant would render this strain more susceptible to mucus-mediated clearance during in vivo nasopharyngeal colonization experiments (12). However, the striking in vivo defect observed in lung and blood models of invasive disease, where the Pcat mutant was completely avirulent, was rather surprising. These data are at odds with a previous study conducted in a serotype 3 strain, which reported that a mutant expressing ∼20% capsule level compared to the wild type was still virulent in the blood (6). However, the disparity between our data may be due to the fact that serotype 3 strains typically express larger amounts of capsule and a simpler polysaccharide structure that is produced via a unique synthesis mechanism (7), coupled with there being a wide range in virulence dependent upon capsular serotypes (52, 53). One potential mechanism for the complete attenuation of the Pcat promoter mutant strain during blood infection may be due to the increase in exposed P-Cho. Previous reports demonstrated that high concentrations of C-reactive protein found in the blood bind exposed P-Cho molecules and initiate the classical complement cascade leading to enhanced clearance (17, 54, 55). Another possibility is that the observed increase of C3 complement deposited on the Pcat mutant likely increases its susceptibility to opsonophagocytic killing (17, 47, 48). Whatever the defect, the phenotype of increased clearance of the Pcat mutant manifests very rapidly, as revealed by the mutant being cleared from the blood by 15 min after intravenous injection (data not shown). In contrast, the complete attenuation of the Pcat mutant in the mouse lung is harder to explain. One possibility is that mechanisms of clearance dependent on C-reactive protein and/or C3 are also happening in the lung. Alternatively, dynamic regulation of capsule is needed for different stages of lung colonization and bacterial multiplication.
The in vivo attenuation of the PtRNAGlu promoter strain, which had comparable levels of capsular polysaccharide compared to TIGR4 in vitro, indicates that some level of dynamic regulation may be needed during colonization and systemic infection. Based on previous work, it is possible to hypothesize that the PtRNAGlu promoter mutant is incapable of effectively colonizing the nasopharynx due to its inability to downregulate capsule and expose underlying surface molecules that facilitate adhesion to the epithelium and subsequent biofilm formation (9, 13, 14). Similarly, if S. pneumoniae needs to augment capsule levels during invasive disease in the lungs or blood (34), the PtRNAGlu promoter mutant may be more sensitive to immune killing due to the presence of constitutively active promoter driving expression of capsule genes. While it is possible that constitutive expression of capsule genes may impact posttranscriptional regulatory mechanisms, we favor the model that the inability to dynamically tune capsule level in various host niches with different capsule requirements results in significant defects during colonization and invasive disease.
The data from experiments using Pcat and PtRNAGlu promoter mutants suggest that the native Pcps promoter possesses some unique property that allows modulation of the capsule level. Our analysis of two chimeric promoter mutant strains, the PcpsCore and PtRNAGluCore mutants, demonstrated that the PcpsCore mutant was capable of colonizing the nasopharynx and causing systemic infection, while the PtRNAGluCore mutant that contained the mutant −10/−35 hexamers was ∼10-fold defective in both niches. These data suggest that the potential regulatory component of the Pcps promoter is located within the core promoter sequences. These data are supported by reports demonstrating that the sequences of the −10/−35 boxes, as well as the sequence and spacing between these two hexamers, can significantly impact transcription of downstream genes (43–45).
In conclusion, this study describes the potential contribution of the native capsular operon promoter, Pcps, on the regulation of capsular polysaccharide levels in S. pneumoniae. These experiments support the model that capsule is a critical virulence factor that must be dynamically controlled in order for S. pneumoniae to thrive in niches in which amounts of capsule are differentially required. Observations from in vivo experiments revealed that a mild reduction in capsular polysaccharide is sufficient to yield a completely avirulent TIGR4 strain. However, a constitutive promoter mutant strain possessing similar initial amounts of capsule compared to the wild-type strain is also attenuated during colonization and invasive disease. Taken together, these data indicate that Pcps and its core elements, in particular, are required for proper modulation of capsule expression to allow optimal colonization and virulence. Future studies are needed to determine if the core promoter sequences, spacing, or both are required for modulating capsular polysaccharide expression and also to determine whether the regulation is graded or stochastic in nature.
FOOTNOTES
- Received 9 October 2013.
- Returned for modification 3 November 2013.
- Accepted 19 November 2013.
- Accepted manuscript posted online 25 November 2013.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
