Mn2+-Dependent Regulation of Multiple Genes in Streptococcus pneumoniae through PsaR and the Resultant Impact on Virulence
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感染与免疫杂志 2006年第2期
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Department of Microbiology, Sanofi Pasteur, 1755 Steeles Avenue West, Toronto, Ontario, Canada M2R 3T42
ABSTRACT
The concentration of Mn2+ is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in Streptococcus pneumoniae, was found to negatively affect the transcription of psaBCA, pcpA, rrgA, rrgB, rrgC, srtBCD, and rlrA in the presence of Mn2+. psaBCA encode an ABC-type transporter for Mn2+. pcpA, rrgA, rrgB, and rrgC encode several outer surface proteins. srtBCD encode a cluster of sortase enzymes, and rlrA encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn2+ concentrations in the wild-type strain and are elevated under both high and low Mn2+ concentrations in a psaR mutant strain. RlrA is an activator of rrgA, rrgB, rrgC, and srtBCD (D. Hava and A. Camilli, Mol. Microbiol. 45:1389-1406, 2002), suggesting that PsaR may indirectly control these genes through rlrA, while PsaR-dependent repression of psaBCA, pcpA, and rlrA transcription is direct. The impact of Mn2+-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of psaR, pcpA, and psaR pcpA mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn2+ acting as a signal for the expression of virulence factors within different host sites.
INTRODUCTION
Streptococcus pneumoniae is primarily an inhabitant of the nasal mucosa of humans, but it can transition from this site to the following internal sites: the lung, causing pneumonia; the eustachian tube, causing otitis media; the blood, causing bacteremia; or the nervous system, causing meningitis. Mn2+ is relatively accessible in the human nasopharynx and has been measured at about 36 μM in saliva, but it is restricted to nanomolar amounts at internal sites (12, 32, 50). Mn2+ could be an important cue by which the immediate environment is sensed and the transitioning to internal body sites is experienced.
The importance of Mn2+ in the cellular physiology of bacteria has only recently been studied at any depth (27, 29). Mn2+ has been shown to have a primary role in the protection from oxidative stress and a variety of roles in metabolism, biosynthesis, and signal transduction (3, 27, 29). Mn2+ is sometimes required for enzymes involved in glycolysis, amino acid metabolism, protein cleavage, nucleic acid degradation, sporulation, germination, and transformation (29). Mn2+ is particularly critical for the lactic acid group of bacteria, many of which have been shown to require Mn2+, but not Fe2+, for survival (3, 13, 37, 41, 59). The use of Mn2+, instead of Fe2+, for the major metal ion needs of the cell eases the challenge of acquiring Fe2+ from within the iron-restricted host environment.
Mn2+ also plays at least a dual role in reducing reactive oxygen species toxicity, including that created by the Fenton reaction, which occurs when Fe2+ interacts with oxygen inside the cell (46). The lactic acid group of bacteria lack catalase but contain superoxide dismutases that require Mn2+ as a cofactor (1, 5, 18, 58), and Mn2+ itself can also act directly to detoxify superoxide and hydrogen peroxide (4-6, 27). We have previously demonstrated that pneumococci require Mn2+ to grow in the presence of Fe2+ under aerobic conditions (31). Mn2+ is used to defend against the toxic effects of superoxide and Fe2+ (31). This makes the capacity to import Mn2+ of particular importance for the abilities of these organisms to handle reactive oxygen species. Most lactic acid bacteria have ATP-binding cassette (ABC)-type Mn2+ transporters, while some have natural resistance-associated macrophage protein family Mn2+ transporters as well (27).
In S. pneumoniae, the Mn2+ transporter is an ABC-type permease encoded by the psaBCA genes. The solute-binding lipoprotein PsaA is a bilobed receptor that initially binds the Zn2+ or Mn2+ metal on the cell surface (15, 34). The transport permease consists of two units of an ATP-binding protein (PsaB) and two units of a hydrophobic membrane protein (PsaC), both of which are also encoded in the operon (Fig. 1A) (42). A gene for thiol peroxidase, PsaD, shares the psaBCAD transcript and is also transcribed separately (42).
The inactivation of any of the genes in the Psa Mn2+ transporter results in profound changes in the virulence potential of bacteria in respiratory tract, otitis media, and sepsis animal models (8, 36) and in a limitation for growth (31, 36). The psa operon is induced as much as 10-fold upon in vivo growth as detected by differential fluorescence screens using a murine respiratory tract infection (RTI) model (36). Expression of psaA was also up-regulated in an intraperitoneal model of infection (43). So, in wild-type pneumococci, the Mn2+ ABC transporter shows increased expression under at least two Mn2+-limited conditions within the host. Additionally, the limited ability of a psaA mutant to grow in an intraperitoneal chamber could be restored upon the addition of Mn2+ (36), indicating the importance of a functional Mn2+ transporter in virulence.
PsaR in S. pneumoniae was previously identified as a homolog of the metal-dependent regulator ScaR of Streptococcus gordonii (30). ScaR represses the sca operon of S. gordonii in response to elevated Mn2+ concentrations (30). Both sca and psa operons encode components of ABC transporters that are capable of bringing Mn2+ into the cell. By analogy, PsaR was considered the most likely candidate for the regulation of the psa operon of S. pneumoniae with Mn2+ as the modulator.
PsaR and ScaR are part of the DtxR family of transcriptional regulators, which respond to metal concentrations and often control transcription at multiple loci (16, 35, 52-54, 56, 57). DtxR itself responds to Fe2+ concentrations (17), and iron-responsive members of the family have been characterized in Corynebacterium diphtheriae (54), Mycobacterium tuberculosis (55), and Staphylococcus epidermidis (25). Other DtxR family members that respond to Mn2+ have been identified in C. diphtheriae, Treponema pallidum, Bacillus subtilis, and Staphylococcus aureus, and they all share similarities to PsaR (2, 26, 47, 48, 51).
To explore the hypothesis that PsaR and/or Mn2+ concentration is a regulator of loci required for growth and virulence in vivo, we constructed an psaR mutant in S. pneumoniae TIGR4 and examined gene expression using RNA slot blot analysis. When the bacteria were grown in a high concentration of Mn2+, transcription of the psa operon was derepressed in the psaR mutant compared to that in the wild type. A number of additional genes were also derepressed, indicating a role for PsaR as a global regulator. The roles of PsaR and one of these regulated gene products, PcpA, were examined in pneumonia and carriage models in mice.
MATERIALS AND METHODS
Bacterial strains, media, and growth conditions. S. pneumoniae strains TIGR4, EF3030, and derivatives thereof were used in this study (Table 1). Pneumococci were grown at 37°C in Todd-Hewitt broth with 0.5% yeast extract (THY) or on blood agar plates unless otherwise indicated. When appropriate, erythromycin was added to the media at a concentration of 0.3 μg/ml.
During strain construction, plasmids were maintained in Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) cells grown in Luria-Bertani (LB) broth or on LB plates with 1.5% agar. Ampicillin (50 μg/ml) for pCR2.1-based plasmids or erythromycin (400 μg/ml) for pJY4164-based plasmids was added to the growth medium.
Divalent metal cations were removed from THY using Chelex-100 (Sigma, St. Louis, MO). Chelex-100 was added at a concentration of 2% (wt/vol) to THY prior to autoclaving. After the Chelex-100-THY mixture was autoclaved, it was stirred overnight at room temperature. The Chelex-100 was allowed to settle out of solution, and the medium was filtered. The resulting medium, designated cTHY, was supplemented with ZnCl2, MgCl2, CaCl2, and FeSO4 to final concentrations of 1 mM each prior to use. MnSO4 was added to concentrations of 0.1 μM or 50 μM.
Strain construction. Insertion-duplication was used to inactivate psaR, rlrA, and pcpA in the parental strain TIGR4 and/or EF3030 (40). Primers used are listed in Table 2. Primer pairs JWJ7 and JWJ8 (psaR), JWJ3 and JWJ4 (rlrA), and JWJ28 and JWJ29 (pcpA) were designed to amplify an internal fragment of the target gene. These fragments were cloned into pCR2.1 using the TOPO TA cloning kit (Invitrogen) and subsequently subcloned into pJY4164 (61). The resulting plasmids were transformed into strain TIGR4 and/or EF3030.
Transformation of pneumococci was performed as described by Yother et al. (62), with modifications. The recipient pneumococcal strain was grown in THY until visible turbidity was present. Bacteria were diluted in competence medium (CTM; THY with 0.2% bovine serum albumin [BSA], 0.2% glucose, and 0.02% CaCl2), and 500 ng/ml of competence-stimulating peptide 1 (CSP-1; EF3030) or CSP-2 (TIGR4) (23) was added to induce competence 14 min prior to the addition of plasmid DNA. The bacteria were incubated at 37°C for 2 h, followed by plating on blood agar containing erythromycin. Resistant transformants were screened for inactivation of the target gene.
An unmarked in-frame deletion of psaR was also constructed in the EF3030 background using a plasmid that contains the regions upstream and downstream of psaR. Primers JWJ86 and JWJ87 were designed to amplify a 1,011-bp region upstream of psaR, and primers JWJ88 and JWJ89 were designed to amplify a 602-bp region downstream of psaR. These fragments were subcloned into pGEM-T (Promega), creating plasmids pJJ062 and pJJ063. The insert in pJJ062 was subcloned into pJY4164 using EcoRI and XbaI sites, creating plasmid pJJ064. The insert in pJJ063 was subcloned into pJJ064 using XbaI and HindIII sites, creating plasmid pJJ066, which was used to transform the psaR mutant JEN9. Transformants were screened for the loss of erythromycin resistance, and the deletion was confirmed by PCR. The deletion mutant was used to make a psaR pcpA double mutant by insertion-duplication of pcpA.
PCR with primer pairs JWJ13 and TT1 (psaR), JWJ10 and TT1 (rlrA), or JWJ30 and TT1 (pcpA) confirmed the integration of the plasmids in the respective correct locations in the pneumococcal genome. Boiled extracts were prepared from each transformant for use as a template in a PCR. Primers JWJ13, JWJ10, and JWJ30 bind upstream of the plasmid integration site, and TT1 is specific for pJY4164. Primers JWJ17 and JWJ89 were used to confirm the deletion of psaR from the pneumococcal genome. This primer pair amplifies a fragment that includes the psaR gene. The mutants constructed were used in the following studies.
RNA extraction and RNA slot blot analysis. RNA samples were extracted using a hot acid phenol protocol (45). Cultures of strains TIGR4 and JEN7 were grown to an optical density at 600 nm of 0.45 in either cTHY with 0.1 μM Mn2+ or cTHY with 50 μM Mn2+. RNA samples were treated with DNase I (Ambion, Inc., Austin, TX). RNA was quantitated by measuring the A260. Digoxigenin (DIG)-labeled probes were generated using gene-specific primers (Table 2) and the PCR DIG probe synthesis kit (Roche Molecular Biochemicals, Indianapolis, IN) following the protocol supplied by the manufacturer. RNA samples were prepared by incubation in sample buffer (50% formamide, 6% formaldehyde, 1x morpholinepropanesulfonic acid [MOPS], pH 7.0) at 60°C for 15 min. The Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA) was used with BrightStar-Plus positively charged nylon membrane (Ambion, Inc.). Samples were loaded in duplicate at concentrations of 4 μg/well and 0.4 μg/well. After application, RNA was cross-linked to the membrane with the UV Stratalinker (Stratagene, La Jolla, CA) and incubated in prehybridization buffer (5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50% formamide, 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate [SDS], and 2% blocking reagent; Roche Molecular Biochemicals) at 45°C for 2 h. Probes were denatured by adding 20 μl to 1 ml of hybridization buffer, boiling for 10 min, followed by a quick chill on ice. Probes were added to the prehybridization buffer, and hybridization was carried out overnight. After removal of the probe, the blot was washed with fresh prehybridization buffer for 30 min at room temperature. Further posthybridization washes were carried out as follows: twice with 2x SSC and 0.1% SDS for 20 min at 37°C, once with 1x SSC and 0.1% SDS for 20 min at 37°C, and once with 0.2x SSC and 0.1% SDS for 45 min at 55°C. Detection was carried out with the DIG DNA detection kit (Roche Molecular Biochemicals) following the protocol supplied by the manufacturer. Blots were exposed to X-ray film (Kodak X-OMAT AR) for 5 to 30 min.
Virulence experiments. Female CBA/CAHN-XID/J (CBA/N) mice, 6 to 12 weeks old, were obtained from Jackson Laboratory (Bar Harbor, ME). The virulence of pneumococcal strains in carriage and pneumonia models was examined as previously described (7, 11, 60). For carriage, mice were inoculated intranasally with 107 CFU of EF3030 and EF3030-derived mutants in a 10-μl volume. For lung infection, mice were anesthetized with isofluorane (Minrad, Inc., Bethlehem, PA) prior to inoculation with pneumococci in a 40-μl volume. TIGR4-derived strains were used for bacteremic invasive disease, while EF3030-derived strains were used for noninvasive disease. For invasive disease, outcome was measured as time to death. For pneumonia and carriage, mice were sacrificed 7 days postinfection. Nasal washes and lung homogenates were prepared for bacterial enumeration as previously described (7, 60). The statistical significance of the difference in median log CFU was analyzed by the Mann-Whitney test (two-tailed test).
Tissue culture. Detroit 562 (D562) human pharyngeal carcinoma cells (ATCC CCL-138; American Type Culture Collection, Manassas, VA) were maintained, passaged, and grown in minimal essential medium (MEM) without L-glutamine, supplemented with Earle's salts and 2.2 g/liter sodium bicarbonate (Gibco, Grand Island, N.Y.), 0.1 mM nonessential amino acids (Gibco), 1.0 mM sodium pyruvate (Gibco), 2.0 mM L-glutamine (Gibco), and 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS) (supplemented MEM). Flat-bottomed 96-well tissue culture-treated plates (Becton Dickinson Labware, Franklin Lakes, NJ) were seeded with approximately 2 x 104 D562 cells/well and cultured at 37°C under 5% CO2 to form confluent monolayers. Monolayers used for the assays were typically 4 to 6 days old.
Adherence assay. Before use, monolayers were washed once with 200 μl of supplemented MEM per well. The medium was aspirated off, and 50 μl of supplemented MEM was added to each well. Frozen stocks of S. pneumoniae TIGR4 and derivatives thereof were removed from –70°C and thawed at room temperature. Serial log10 dilutions of bacterial stocks were prepared in supplemented MEM to a final volume of 2.0 ml in 6.0-ml polypropylene tubes. To obtain an estimate of CFU added per well, each strain was further diluted 10-fold in Dulbecco's phosphate-buffered saline (PBS) (Gibco) supplemented with 0.2% bovine serum albumin (Sigma-Aldrich) to a final volume of 100 μl in 96-well round-bottomed non-tissue-culture-treated plates (Becton Dickinson Labware). Each sample dilution was prepared in duplicate. A 40-μl volume of each diluted sample was plated onto Trypticase soy agar (TSA II) plates containing 5% sheep blood (Becton Dickinson Biosciences, Cockeysville, MD) and incubated overnight at 37°C under 5% CO2. Each bacterial strain was tested at 4 to 5 dilution points within a range of 1 x 103 CFU added/well to 2 x 108 CFU added/well.
For adherence assay plates, 50-μl samples of appropriately diluted bacteria in supplemented MEM were added to triplicate wells of a 96-well plate containing washed D562 monolayers. After a 2.5-h incubation at 37°C under 5% CO2, monolayers were washed five times with 200 μl/well of PBS supplemented with 0.2% BSA to remove nonadherent bacteria. D562 cells were detached by the addition of 20 μl/well of 0.05% trypsin with 0.53 mM EDTA-Na4 (Gibco). After 10 min at 37°C, each sample well was neutralized with 80 μl supplemented MEM, and samples were mixed by pipetting up and down 50 times with a Biohit Proline electronic pipettor (Biohit, Helsinki, Finland) set to a 55-μl volume and maximum speed mixing mode. Tenfold plating dilutions of each sample were prepared in PBS with 0.2% BSA to a final volume of 100 μl in 96-well round-bottomed non-tissue-culture-treated plates (Becton Dickinson Labware). A volume of 40 μl of each diluted sample was plated onto TSA II agar plates containing 5% sheep blood. Agar plates were incubated 18 to 36 h at 37°C under 5% CO2. CFU per plate were counted under magnification. The average CFU count of replicate plates was used to estimate the number of CFU added per well and the number of CFU bound per well.
RESULTS
Identification of PsaR-regulated genes by RNA slot blot analysis. RNA slot blot analysis was used to compare the expression of psaBCA in S. pneumoniae TIGR4 and the psaR mutant. RNA was extracted from TIGR4 and the psaR mutant that had been grown in either cTHY with 0.1 μM Mn2+ or cTHY with 50 μM Mn2+. DIG-labeled probes were generated with gene-specific primers, using TIGR4 genomic DNA as a template. A probe for ldh was used to confirm that equal amounts of RNA were added to each well. After development of the blot, unsaturated bands were measured by densitometry. In all cases, duplicate samples varied by less than 20%. The mean for the duplicate samples was used in the calculation of ratios for relative expression (Fig. 2A and B).
When RNA from strain TIGR4 grown in 0.1 μM Mn2+ was compared to RNA from TIGR4 grown in 50 μM Mn2+, psaB, psaC, and psaA were all expressed under low Mn2+ conditions; however, expression was diminished when grown under high Mn2+ conditions (Fig. 2A and C). Expression of psaB, psaC, and psaA was increased in the psaR mutant strain than in TIGR4 when both were grown in 50 μM Mn2+ (Fig. 2B and C). This demonstrates that psaB, psaC, and psaA are repressed by PsaR in response to elevated Mn2+ concentrations. No regulation was seen for psaD (data not shown) however, implying that the P2 promoter may be responsible for most of the transcription of psaD and that its transcription is independent of PsaR, consistent with the results of McAllister et al. (38).
In addition to the psa operon, five other potential virulence factors were found to be differentially expressed in a psaR mutant strain in a preliminary microarray expression profile screen (data not shown). The pcpA, rrgA, rrgB, rrgC, and srtB genes were tested for PsaR- and Mn2+-dependent regulation by RNA slot blot analysis. All were found to be expressed under the low Mn2+ conditions but repressed under high Mn2+ conditions in the wild-type strain (Fig. 2A) and to be induced in the psaR mutant under both growth conditions (Fig. 2B and C). These genes encode putative surface proteins with unknown functions (pcpA, rrgA, rrgB, and rrgC) and a putative sortase (srtB). This set of genes is found to be induced under growth in low Mn2+ (0.1 μM) in the wild-type strain and also when the PsaR regulator is inactivated. Taken together, these findings suggest that Mn2+ acts with PsaR to cause the observed repression of this regulon. Mn2+ was the only variable in these experiments. Because Fe2+, Mg2+, Ca2+, and Zn2+ concentrations were nonlimiting and were kept constant in all our experiments, it is unlikely that these cations affected the PsaR-dependent regulation observed here. Inactivation of psaR was not judged as likely to create any gene expression change through a polar effect, since the gene immediately downstream of psaR is a transposase that is convergently transcribed with psaR.
PsaR affects transcription of multiple genes via expression of rlrA. Several of the genes under study, rrgA, rrgB, rrgC, and srtB, are clustered in the genome and located near the putative regulator, rlrA (Fig. 1B). Hava et al. demonstrated that RlrA activates the transcription of rrgA, rrgB, rrgC, and srtB (21). To confirm that this gene cluster is regulated by RlrA, we constructed an rlrA mutant strain of TIGR4. The expression of rrgA, rrgB, rrgC, and srtB in the wild-type TIGR4 strain and the rlrA mutant was examined. All four genes were expressed at almost undetectable levels in the rlrA mutant strain, confirming the results of Hava et al. (results not shown) (21). This raised the possibility that PsaR influences the expression of RlrA-regulated genes by repressing transcription of rlrA. When examined by slot blot analysis, the expression of rlrA was lower in the wild-type TIGR4 than in the psaR mutant under conditions of high Mn2+ (Fig. 2B and C). This indicated that PsaR affected transcription of rlrA, but the magnitude of the change in expression (twofold) was significantly less than in other PsaR-regulated genes. Thus, PsaR may be affecting the transcription of rrgA, rrgB, rrgC, and srtB through its effect on the expression of rlrA. Since RlrA is a transcriptional activator, a minimal increase in its expression can lead to a greater increase in the expression of its target genes. Thus, the relief of repression of the activator leads to an increase in the expression of rrgA, rrgB, rrgC, and srtB. The unlinked pcpA gene exhibited increased expression in the psaR mutant strain but was not affected in the rlrA mutant strain (data not shown), indicating that pcpA expression is not influenced by RlrA.
Inactivation of psaR and pcpA differentially impact carriage and pneumonia. Because PsaR represses the Mn2+ transporter psaABC and because Mn2+ levels are believed to be much higher in the nasopharynx than in the lung, we examined the effects of psaR mutations on virulence in murine models for pneumonia and nasopharyngeal carriage. Intranasal infection with strain EF3030 results in carriage and/or pneumonia depending on the infection protocol but does not progress to bacteremia and sepsis at the doses used in this study (11). For this purpose, psaR, pcpA, and psaR pcpA strains were constructed in the EF3030 background as described in Materials and Methods. EF3030 was chosen because the mouse pneumonia model is most like pneumonia in humans for this strain (11). The EF3030 strain lacks the rlrA-regulated gene cluster, so these results are relevant only to the other PsaR-regulated genes. Slot blot analysis confirmed that PsaR-mediated regulation of the psa operon and pcpA occurs in EF3030 (results not shown).
A reasonable prediction for the Mn2+-dependent repression was that the greatest impact of inactivation of psaR would be observed in the higher Mn2+ environment of the nasopharynx. This is because it is under high Mn2+ that regulated gene expression varies. To determine whether the mutant strains were deficient in the ability to establish nasopharyngeal carriage, we examined the colonization of each of the mutants compared with EF3030 following intranasal infection with 106 CFU. The bacteria were given in a 10-μl volume to nonanesthetized mice to produce carriage without pneumonia (11). In this infection model, no significant difference in nasopharyngeal colonization was seen between the mutants and the wild-type strains (Fig. 3). These results suggest that neither psaR nor pcpA is required for the establishment of carriage of pneumococci.
To determine the effects of the inactivation of psaR and pcpA in pneumonia, mice were inoculated with 107 CFU in a 40-μl volume while anesthetized to ensure aspiration of pneumococci. Each of the mutant strains showed attenuated virulence in the lung, with a significant decrease in the number of bacterial CFU/lung compared to that of EF3030 (Fig. 4) (P 0.0001 for each). The results for the pcpA mutant strain support a role for PcpA in bacterial persistence within the lung. There was clear attenuation in the virulence of the pcpA mutant strain. The attenuated growth of the psaR mutant strain in the lung is puzzling. If the lung were subject to lower Mn2+ conditions than the nasopharynx, then PsaR repression would be relieved, and there would be little phenotypic differences between the mutant and wild-type strains. The psaR mutant strain should be expressing the Psa transporter encoded by psaABC, as well as the surface protein PcpA at wild-type levels in EF3030 (Fig. 2C). The absence of any of these PsaR-regulated proteins, as judged by results with knockout mutants, attenuates bacterial growth in the lung (20, 21). It is possible that the mucosal surfaces in the lung contain some levels of Mn2+, allowing for some PsaR-mediated repression to occur (that is relieved in the mutant strain). Alternatively, it is possible that the attenuation of the psaR mutant strain in the lung may be a consequence of overexpression of one or more of the regulated gene products. The psaR pcpA double mutant was slightly more attenuated than either single mutant strain, suggesting that there are two separate phenotypic impacts in the single mutant strains.
Inactivation of psaR does not affect adherence. It has previously been hypothesized that the adherence defect observed in psaA mutants may be due to the disruption of the activity of an Mn2+-dependent transcriptional regulator (44). To test this hypothesis, the adherence of TIGR4 and its psaR mutant to a nasopharyngeal cell line was examined. The psaA mutant strain previously shown to have altered adherence in this assay (31) was included as a control. Binding curves were generated for each strain, and the psaR mutant was found to adhere to the D562 cells at wild-type levels throughout the curve, while the psaA mutant exhibited a reduction in adherence (Fig. 5). These data indicate that PsaR is not responsible for the regulation of the adherence function that is defective in psaA mutants, but this does not eliminate the possibility that Mn2+ may be involved in the regulation of adherence.
DISCUSSION
The control of Mn2+ homeostasis is expected to be important to the bacterial cell, and PsaR is important to this control in pneumococci. On the basis of our analysis of transcription patterns, PsaR appears to function as a repressor when sufficient Mn2+ is present. PsaR has a predicted metal binding site and is likely to bind to its targets when Mn2+ is present. PsaR shares 76% similarity and 65% amino acid sequence identity with ScaR, the Mn2+-dependent repressor of S. gordonii (30). The target sequence for ScaR binding has been identified, and the binding site for ScaR is 90% identical to a putative PsaR operator located in the promoter region of the psa operon (30). Additionally, the residues responsible for the selective binding of Mn2+ or Fe2+ by MntR or DtxR, respectively, have been previously identified (19), and the sequence of PsaR suggests it would bind Mn2+. This mode of action for PsaR was strongly supported by our data: inactivation of PsaR resulted in the derepression of a set of genes that are also derepressed when low Mn2+ concentrations are present in the wild-type strain.
PsaR-regulated genes were located in three different regions of the chromosome, indicating that PsaR is a multigene regulator. A model for PsaR regulation is presented in Fig. 6. In this model, the DNA-binding activity of PsaR is activated by the binding of Mn2+, which then represses the transcription of psaBCA, pcpA, and rlrA. Repression of rlrA leads to lower expression of rrgA, rrgBC, and srtBCD, since these genes are activated by RlrA. The potential regulatory cascade with PsaR and RlrA may explain the effect that psaR inactivation and Mn2+ concentration have on the expression of rrgA, rrgB, rrgC, and srtBCD. This regulatory cascade is evidenced by a greater change of expression in RlrA-regulated genes than in rlrA itself in comparing expression between psaR and TIGR4. In essence, the cascade allows the rlrA regulon to respond to the external Mn2+ concentration.
As an essential trace element, Mn2+ is extremely limited within the human body, where it may be complexed with carrier proteins (50). The highest concentration of in vivo Mn2+ appears to be in saliva, where it is 36 μM (12, 32). Thus, while pneumococci are in the nasopharynx, Mn2+ is presumably relatively abundant and expression of psaBCA, pcpA, and the rlrA-controlled genes would be repressed. When pneumococci invade the lung or bloodstream, the levels of Mn2+ are much lower (20 nM given for serum/plasma) (29, 50). Expression of the psa operon would be increased, inducing the high-affinity transporter in order to obtain Mn2+ that the bacterium needs to survive. Consistent with this model, transcript levels of psaA were increased in bacteria recovered from blood of an infected mouse compared to bacteria grown in vitro (43). Also, the repeated recovery of psaA, psaB, or psaC in a screen for in vivo-induced genes in a murine respiratory tract infection model is consistent with this scenario (36). The external change in Mn2+ concentration is clearly one factor that signals the bacterium to change its expression of virulence factors in response to its environment.
We identified additional genes outside of the psa transporter itself that are repressed by PsaR in response to Mn2+ concentrations. These include pcpA, rrgA, rrgB, rrgC, and srtB. PcpA is a choline-binding protein that contains leucine-rich repeats (49), which can often be involved in protein-protein interactions (33). On the basis of this, it was hypothesized that PcpA functions as an adhesin (49). Signature-tagged mutagenesis identified pcpA as being important for both pneumonia and bacteremia (20). In our study, the pcpA mutant was able to colonize the nasopharynx similar to the wild type, but Hava and Camilli found that a pcpA mutant was less effective in colonization when studied using a competitive carriage model (20). Our data supports a role for PcpA in the persistent establishment of growth during pneumonia, even in the absence of competition with the wild-type strain.
RrgA, RrgB, and RrgC are putative surface proteins that contain YPXTG, IPXTG, and VPXTG motifs, respectively, at their carboxy-terminal ends. These putative recognition sites are predicted to be recognized by sortase enzymes, which would then anchor these proteins to the cell wall. The rrgABC genes are clustered with the srtBCD operon on the chromosome (Fig. 1B). The proximity of the putative sortases to rrgA, rrgB, and rrgC and the unusual sorting motifs suggest that the sortases SrtB, SrtC, and/or SrtD specifically anchor these gene products. No specific binding activities for the Rrg proteins have been reported, although their weak homology to some microbial surface components recognizing adhesive matrix molecules, which often bind to fibronectin or fibrinogen, has suggested a possible adhesive function (9, 20). All of the genes in this cluster are positively controlled by rlrA (21). The rlrA-controlled region has an atypical GC content relative to the rest of the genome, and it is not common to all pneumococcal strains, indicating the possibility of horizontal transfer (28, 58). In the published studies, rrgA and srtD mutants showed reduced virulence in competitive pneumonia models of infection (20-22). Additionally, rrgC and srtC mutants exhibited an increased virulence (higher competitive index) for colonization in a carriage model, although rrgC did not reach significance (20). All of the rlrA islet genes are up-regulated in the TIGR4 psaR mutant strain. In the EF3030 psaR strain, psaABC and pcpA are similarly up-regulated. However, the genes in the rlrA islet are not present in this strain background.
Strain EF3030 is used in the mouse pneumonia model, because it is rapidly cleared from the blood, it causes pneumonia that is verifiable by histology, and there is an absence of bacteremia. This is similar to the natural disease state in humans (11). The net effect of both the pcpA mutation and the psaR mutation in this model was an attenuation of virulence. As EF3030 lacks the rlrA cluster, in this strain the major impact of the psaR mutation is likely to be felt through the elevated expression of either PcpA or the Psa transporter. Alternatively, it is possible that additional genes not found in our in vitro system may be regulated by psaR in the in vivo setting, contributing to the phenotype.
The expression of pcpA was also regulated in response to Mn2+ in our work, leading to a predicted lower level of expression in the nasopharynx and a higher level of expression in the lung and blood. Because PcpA did not appear to be required for colonization of the nasopharynx in our carriage model (Fig. 3, pcpA mutant), we predict that it may play its major role in the lung. This would be consistent with its regulation, since pcpA is repressed by PsaR under the Mn2+ conditions similar to those in the nasopharynx. The unregulated expression of PsaR-regulated proteins also did not reduce colonization, judged by the similar CFU loads in the wild-type and psaR mutant strains. Together, these observations suggest that the major impact of regulation in response to Mn2+ is felt in the pneumonia model. Our data imply that both too little surface protein in the pcpA mutant and too much surface protein in the psaR mutant may be detrimental.
These results highlight the complexity involved in understanding the nature of the phenotype of any dysregulation. Strain EF3030 lacked the rlrA cluster, but in another pneumococcal strain, the impact of a psaR mutation or the Mn2+ concentration may be felt through the rlrA cluster. Hemsley et al. have found that the MgrA regulator acts as a repressor on RlrA, but mgrA mutations also result in the attenuation of virulence in pneumonia (24). Results suggested that although some rlrA-regulated products may be required for full virulence in pneumonia, the elevated levels of one or more of these gene products that occurred under the loss of negative control by mgrA was also detrimental to the development of full virulence. A similar scenario may occur for PsaR regulation, either through PcpA or through the rlrA cluster in strains containing it.
The psaBCA operon has been shown to be activated by a two-component system, RR04/HK04, in strain TIGR4 but this did not occur in strain D39 and 0011389 (39). RR04 appeared to interact separately with the psa operon, rather than indirectly through psaR, based on the observation that psaR transcription was unaltered in the RR04 mutant (39) and that other PsaR-regulated genes, pcpA or the rlrA regulon, were not similarly affected. This regulatory system may put the expression of the psa operon under the control of an environmental signal in addition to Mn2+; but this appears to be specific to TIGR4.
This is the first demonstration of a pathogen using Mn2+ as a signal to modulate the expression of virulence genes which may have different impacts in different host sites. The disruption of this regulation negatively impacts virulence in a pneumonia model. We have also demonstrated that PcpA, a PsaR-regulated gene, has a role in the persistence of pneumococci in pneumonia but does not have a role in colonization of the nasopharynx. The control of expression of additional PsaR-regulated genes involves multiple factors, and more work is necessary to determine how these regulators interact to control the expression of multiple pneumococcal virulence factors.
ACKNOWLEDGMENTS
These studies were conducted with the support from NIH grants AI053749, AI40645, and AI21548. Jason Johnston was supported by NIAID training grant T32 AI 07041.
We thank Martina Ochs for her input in reviewing the manuscript prior to publication. We thank Yvette Hale for her assistance with the virulence studies. Additionally, we thank Priya Balachandran for help with the RNA procedures.
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Department of Microbiology, Sanofi Pasteur, 1755 Steeles Avenue West, Toronto, Ontario, Canada M2R 3T42
ABSTRACT
The concentration of Mn2+ is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in Streptococcus pneumoniae, was found to negatively affect the transcription of psaBCA, pcpA, rrgA, rrgB, rrgC, srtBCD, and rlrA in the presence of Mn2+. psaBCA encode an ABC-type transporter for Mn2+. pcpA, rrgA, rrgB, and rrgC encode several outer surface proteins. srtBCD encode a cluster of sortase enzymes, and rlrA encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn2+ concentrations in the wild-type strain and are elevated under both high and low Mn2+ concentrations in a psaR mutant strain. RlrA is an activator of rrgA, rrgB, rrgC, and srtBCD (D. Hava and A. Camilli, Mol. Microbiol. 45:1389-1406, 2002), suggesting that PsaR may indirectly control these genes through rlrA, while PsaR-dependent repression of psaBCA, pcpA, and rlrA transcription is direct. The impact of Mn2+-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of psaR, pcpA, and psaR pcpA mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn2+ acting as a signal for the expression of virulence factors within different host sites.
INTRODUCTION
Streptococcus pneumoniae is primarily an inhabitant of the nasal mucosa of humans, but it can transition from this site to the following internal sites: the lung, causing pneumonia; the eustachian tube, causing otitis media; the blood, causing bacteremia; or the nervous system, causing meningitis. Mn2+ is relatively accessible in the human nasopharynx and has been measured at about 36 μM in saliva, but it is restricted to nanomolar amounts at internal sites (12, 32, 50). Mn2+ could be an important cue by which the immediate environment is sensed and the transitioning to internal body sites is experienced.
The importance of Mn2+ in the cellular physiology of bacteria has only recently been studied at any depth (27, 29). Mn2+ has been shown to have a primary role in the protection from oxidative stress and a variety of roles in metabolism, biosynthesis, and signal transduction (3, 27, 29). Mn2+ is sometimes required for enzymes involved in glycolysis, amino acid metabolism, protein cleavage, nucleic acid degradation, sporulation, germination, and transformation (29). Mn2+ is particularly critical for the lactic acid group of bacteria, many of which have been shown to require Mn2+, but not Fe2+, for survival (3, 13, 37, 41, 59). The use of Mn2+, instead of Fe2+, for the major metal ion needs of the cell eases the challenge of acquiring Fe2+ from within the iron-restricted host environment.
Mn2+ also plays at least a dual role in reducing reactive oxygen species toxicity, including that created by the Fenton reaction, which occurs when Fe2+ interacts with oxygen inside the cell (46). The lactic acid group of bacteria lack catalase but contain superoxide dismutases that require Mn2+ as a cofactor (1, 5, 18, 58), and Mn2+ itself can also act directly to detoxify superoxide and hydrogen peroxide (4-6, 27). We have previously demonstrated that pneumococci require Mn2+ to grow in the presence of Fe2+ under aerobic conditions (31). Mn2+ is used to defend against the toxic effects of superoxide and Fe2+ (31). This makes the capacity to import Mn2+ of particular importance for the abilities of these organisms to handle reactive oxygen species. Most lactic acid bacteria have ATP-binding cassette (ABC)-type Mn2+ transporters, while some have natural resistance-associated macrophage protein family Mn2+ transporters as well (27).
In S. pneumoniae, the Mn2+ transporter is an ABC-type permease encoded by the psaBCA genes. The solute-binding lipoprotein PsaA is a bilobed receptor that initially binds the Zn2+ or Mn2+ metal on the cell surface (15, 34). The transport permease consists of two units of an ATP-binding protein (PsaB) and two units of a hydrophobic membrane protein (PsaC), both of which are also encoded in the operon (Fig. 1A) (42). A gene for thiol peroxidase, PsaD, shares the psaBCAD transcript and is also transcribed separately (42).
The inactivation of any of the genes in the Psa Mn2+ transporter results in profound changes in the virulence potential of bacteria in respiratory tract, otitis media, and sepsis animal models (8, 36) and in a limitation for growth (31, 36). The psa operon is induced as much as 10-fold upon in vivo growth as detected by differential fluorescence screens using a murine respiratory tract infection (RTI) model (36). Expression of psaA was also up-regulated in an intraperitoneal model of infection (43). So, in wild-type pneumococci, the Mn2+ ABC transporter shows increased expression under at least two Mn2+-limited conditions within the host. Additionally, the limited ability of a psaA mutant to grow in an intraperitoneal chamber could be restored upon the addition of Mn2+ (36), indicating the importance of a functional Mn2+ transporter in virulence.
PsaR in S. pneumoniae was previously identified as a homolog of the metal-dependent regulator ScaR of Streptococcus gordonii (30). ScaR represses the sca operon of S. gordonii in response to elevated Mn2+ concentrations (30). Both sca and psa operons encode components of ABC transporters that are capable of bringing Mn2+ into the cell. By analogy, PsaR was considered the most likely candidate for the regulation of the psa operon of S. pneumoniae with Mn2+ as the modulator.
PsaR and ScaR are part of the DtxR family of transcriptional regulators, which respond to metal concentrations and often control transcription at multiple loci (16, 35, 52-54, 56, 57). DtxR itself responds to Fe2+ concentrations (17), and iron-responsive members of the family have been characterized in Corynebacterium diphtheriae (54), Mycobacterium tuberculosis (55), and Staphylococcus epidermidis (25). Other DtxR family members that respond to Mn2+ have been identified in C. diphtheriae, Treponema pallidum, Bacillus subtilis, and Staphylococcus aureus, and they all share similarities to PsaR (2, 26, 47, 48, 51).
To explore the hypothesis that PsaR and/or Mn2+ concentration is a regulator of loci required for growth and virulence in vivo, we constructed an psaR mutant in S. pneumoniae TIGR4 and examined gene expression using RNA slot blot analysis. When the bacteria were grown in a high concentration of Mn2+, transcription of the psa operon was derepressed in the psaR mutant compared to that in the wild type. A number of additional genes were also derepressed, indicating a role for PsaR as a global regulator. The roles of PsaR and one of these regulated gene products, PcpA, were examined in pneumonia and carriage models in mice.
MATERIALS AND METHODS
Bacterial strains, media, and growth conditions. S. pneumoniae strains TIGR4, EF3030, and derivatives thereof were used in this study (Table 1). Pneumococci were grown at 37°C in Todd-Hewitt broth with 0.5% yeast extract (THY) or on blood agar plates unless otherwise indicated. When appropriate, erythromycin was added to the media at a concentration of 0.3 μg/ml.
During strain construction, plasmids were maintained in Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) cells grown in Luria-Bertani (LB) broth or on LB plates with 1.5% agar. Ampicillin (50 μg/ml) for pCR2.1-based plasmids or erythromycin (400 μg/ml) for pJY4164-based plasmids was added to the growth medium.
Divalent metal cations were removed from THY using Chelex-100 (Sigma, St. Louis, MO). Chelex-100 was added at a concentration of 2% (wt/vol) to THY prior to autoclaving. After the Chelex-100-THY mixture was autoclaved, it was stirred overnight at room temperature. The Chelex-100 was allowed to settle out of solution, and the medium was filtered. The resulting medium, designated cTHY, was supplemented with ZnCl2, MgCl2, CaCl2, and FeSO4 to final concentrations of 1 mM each prior to use. MnSO4 was added to concentrations of 0.1 μM or 50 μM.
Strain construction. Insertion-duplication was used to inactivate psaR, rlrA, and pcpA in the parental strain TIGR4 and/or EF3030 (40). Primers used are listed in Table 2. Primer pairs JWJ7 and JWJ8 (psaR), JWJ3 and JWJ4 (rlrA), and JWJ28 and JWJ29 (pcpA) were designed to amplify an internal fragment of the target gene. These fragments were cloned into pCR2.1 using the TOPO TA cloning kit (Invitrogen) and subsequently subcloned into pJY4164 (61). The resulting plasmids were transformed into strain TIGR4 and/or EF3030.
Transformation of pneumococci was performed as described by Yother et al. (62), with modifications. The recipient pneumococcal strain was grown in THY until visible turbidity was present. Bacteria were diluted in competence medium (CTM; THY with 0.2% bovine serum albumin [BSA], 0.2% glucose, and 0.02% CaCl2), and 500 ng/ml of competence-stimulating peptide 1 (CSP-1; EF3030) or CSP-2 (TIGR4) (23) was added to induce competence 14 min prior to the addition of plasmid DNA. The bacteria were incubated at 37°C for 2 h, followed by plating on blood agar containing erythromycin. Resistant transformants were screened for inactivation of the target gene.
An unmarked in-frame deletion of psaR was also constructed in the EF3030 background using a plasmid that contains the regions upstream and downstream of psaR. Primers JWJ86 and JWJ87 were designed to amplify a 1,011-bp region upstream of psaR, and primers JWJ88 and JWJ89 were designed to amplify a 602-bp region downstream of psaR. These fragments were subcloned into pGEM-T (Promega), creating plasmids pJJ062 and pJJ063. The insert in pJJ062 was subcloned into pJY4164 using EcoRI and XbaI sites, creating plasmid pJJ064. The insert in pJJ063 was subcloned into pJJ064 using XbaI and HindIII sites, creating plasmid pJJ066, which was used to transform the psaR mutant JEN9. Transformants were screened for the loss of erythromycin resistance, and the deletion was confirmed by PCR. The deletion mutant was used to make a psaR pcpA double mutant by insertion-duplication of pcpA.
PCR with primer pairs JWJ13 and TT1 (psaR), JWJ10 and TT1 (rlrA), or JWJ30 and TT1 (pcpA) confirmed the integration of the plasmids in the respective correct locations in the pneumococcal genome. Boiled extracts were prepared from each transformant for use as a template in a PCR. Primers JWJ13, JWJ10, and JWJ30 bind upstream of the plasmid integration site, and TT1 is specific for pJY4164. Primers JWJ17 and JWJ89 were used to confirm the deletion of psaR from the pneumococcal genome. This primer pair amplifies a fragment that includes the psaR gene. The mutants constructed were used in the following studies.
RNA extraction and RNA slot blot analysis. RNA samples were extracted using a hot acid phenol protocol (45). Cultures of strains TIGR4 and JEN7 were grown to an optical density at 600 nm of 0.45 in either cTHY with 0.1 μM Mn2+ or cTHY with 50 μM Mn2+. RNA samples were treated with DNase I (Ambion, Inc., Austin, TX). RNA was quantitated by measuring the A260. Digoxigenin (DIG)-labeled probes were generated using gene-specific primers (Table 2) and the PCR DIG probe synthesis kit (Roche Molecular Biochemicals, Indianapolis, IN) following the protocol supplied by the manufacturer. RNA samples were prepared by incubation in sample buffer (50% formamide, 6% formaldehyde, 1x morpholinepropanesulfonic acid [MOPS], pH 7.0) at 60°C for 15 min. The Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA) was used with BrightStar-Plus positively charged nylon membrane (Ambion, Inc.). Samples were loaded in duplicate at concentrations of 4 μg/well and 0.4 μg/well. After application, RNA was cross-linked to the membrane with the UV Stratalinker (Stratagene, La Jolla, CA) and incubated in prehybridization buffer (5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50% formamide, 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate [SDS], and 2% blocking reagent; Roche Molecular Biochemicals) at 45°C for 2 h. Probes were denatured by adding 20 μl to 1 ml of hybridization buffer, boiling for 10 min, followed by a quick chill on ice. Probes were added to the prehybridization buffer, and hybridization was carried out overnight. After removal of the probe, the blot was washed with fresh prehybridization buffer for 30 min at room temperature. Further posthybridization washes were carried out as follows: twice with 2x SSC and 0.1% SDS for 20 min at 37°C, once with 1x SSC and 0.1% SDS for 20 min at 37°C, and once with 0.2x SSC and 0.1% SDS for 45 min at 55°C. Detection was carried out with the DIG DNA detection kit (Roche Molecular Biochemicals) following the protocol supplied by the manufacturer. Blots were exposed to X-ray film (Kodak X-OMAT AR) for 5 to 30 min.
Virulence experiments. Female CBA/CAHN-XID/J (CBA/N) mice, 6 to 12 weeks old, were obtained from Jackson Laboratory (Bar Harbor, ME). The virulence of pneumococcal strains in carriage and pneumonia models was examined as previously described (7, 11, 60). For carriage, mice were inoculated intranasally with 107 CFU of EF3030 and EF3030-derived mutants in a 10-μl volume. For lung infection, mice were anesthetized with isofluorane (Minrad, Inc., Bethlehem, PA) prior to inoculation with pneumococci in a 40-μl volume. TIGR4-derived strains were used for bacteremic invasive disease, while EF3030-derived strains were used for noninvasive disease. For invasive disease, outcome was measured as time to death. For pneumonia and carriage, mice were sacrificed 7 days postinfection. Nasal washes and lung homogenates were prepared for bacterial enumeration as previously described (7, 60). The statistical significance of the difference in median log CFU was analyzed by the Mann-Whitney test (two-tailed test).
Tissue culture. Detroit 562 (D562) human pharyngeal carcinoma cells (ATCC CCL-138; American Type Culture Collection, Manassas, VA) were maintained, passaged, and grown in minimal essential medium (MEM) without L-glutamine, supplemented with Earle's salts and 2.2 g/liter sodium bicarbonate (Gibco, Grand Island, N.Y.), 0.1 mM nonessential amino acids (Gibco), 1.0 mM sodium pyruvate (Gibco), 2.0 mM L-glutamine (Gibco), and 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS) (supplemented MEM). Flat-bottomed 96-well tissue culture-treated plates (Becton Dickinson Labware, Franklin Lakes, NJ) were seeded with approximately 2 x 104 D562 cells/well and cultured at 37°C under 5% CO2 to form confluent monolayers. Monolayers used for the assays were typically 4 to 6 days old.
Adherence assay. Before use, monolayers were washed once with 200 μl of supplemented MEM per well. The medium was aspirated off, and 50 μl of supplemented MEM was added to each well. Frozen stocks of S. pneumoniae TIGR4 and derivatives thereof were removed from –70°C and thawed at room temperature. Serial log10 dilutions of bacterial stocks were prepared in supplemented MEM to a final volume of 2.0 ml in 6.0-ml polypropylene tubes. To obtain an estimate of CFU added per well, each strain was further diluted 10-fold in Dulbecco's phosphate-buffered saline (PBS) (Gibco) supplemented with 0.2% bovine serum albumin (Sigma-Aldrich) to a final volume of 100 μl in 96-well round-bottomed non-tissue-culture-treated plates (Becton Dickinson Labware). Each sample dilution was prepared in duplicate. A 40-μl volume of each diluted sample was plated onto Trypticase soy agar (TSA II) plates containing 5% sheep blood (Becton Dickinson Biosciences, Cockeysville, MD) and incubated overnight at 37°C under 5% CO2. Each bacterial strain was tested at 4 to 5 dilution points within a range of 1 x 103 CFU added/well to 2 x 108 CFU added/well.
For adherence assay plates, 50-μl samples of appropriately diluted bacteria in supplemented MEM were added to triplicate wells of a 96-well plate containing washed D562 monolayers. After a 2.5-h incubation at 37°C under 5% CO2, monolayers were washed five times with 200 μl/well of PBS supplemented with 0.2% BSA to remove nonadherent bacteria. D562 cells were detached by the addition of 20 μl/well of 0.05% trypsin with 0.53 mM EDTA-Na4 (Gibco). After 10 min at 37°C, each sample well was neutralized with 80 μl supplemented MEM, and samples were mixed by pipetting up and down 50 times with a Biohit Proline electronic pipettor (Biohit, Helsinki, Finland) set to a 55-μl volume and maximum speed mixing mode. Tenfold plating dilutions of each sample were prepared in PBS with 0.2% BSA to a final volume of 100 μl in 96-well round-bottomed non-tissue-culture-treated plates (Becton Dickinson Labware). A volume of 40 μl of each diluted sample was plated onto TSA II agar plates containing 5% sheep blood. Agar plates were incubated 18 to 36 h at 37°C under 5% CO2. CFU per plate were counted under magnification. The average CFU count of replicate plates was used to estimate the number of CFU added per well and the number of CFU bound per well.
RESULTS
Identification of PsaR-regulated genes by RNA slot blot analysis. RNA slot blot analysis was used to compare the expression of psaBCA in S. pneumoniae TIGR4 and the psaR mutant. RNA was extracted from TIGR4 and the psaR mutant that had been grown in either cTHY with 0.1 μM Mn2+ or cTHY with 50 μM Mn2+. DIG-labeled probes were generated with gene-specific primers, using TIGR4 genomic DNA as a template. A probe for ldh was used to confirm that equal amounts of RNA were added to each well. After development of the blot, unsaturated bands were measured by densitometry. In all cases, duplicate samples varied by less than 20%. The mean for the duplicate samples was used in the calculation of ratios for relative expression (Fig. 2A and B).
When RNA from strain TIGR4 grown in 0.1 μM Mn2+ was compared to RNA from TIGR4 grown in 50 μM Mn2+, psaB, psaC, and psaA were all expressed under low Mn2+ conditions; however, expression was diminished when grown under high Mn2+ conditions (Fig. 2A and C). Expression of psaB, psaC, and psaA was increased in the psaR mutant strain than in TIGR4 when both were grown in 50 μM Mn2+ (Fig. 2B and C). This demonstrates that psaB, psaC, and psaA are repressed by PsaR in response to elevated Mn2+ concentrations. No regulation was seen for psaD (data not shown) however, implying that the P2 promoter may be responsible for most of the transcription of psaD and that its transcription is independent of PsaR, consistent with the results of McAllister et al. (38).
In addition to the psa operon, five other potential virulence factors were found to be differentially expressed in a psaR mutant strain in a preliminary microarray expression profile screen (data not shown). The pcpA, rrgA, rrgB, rrgC, and srtB genes were tested for PsaR- and Mn2+-dependent regulation by RNA slot blot analysis. All were found to be expressed under the low Mn2+ conditions but repressed under high Mn2+ conditions in the wild-type strain (Fig. 2A) and to be induced in the psaR mutant under both growth conditions (Fig. 2B and C). These genes encode putative surface proteins with unknown functions (pcpA, rrgA, rrgB, and rrgC) and a putative sortase (srtB). This set of genes is found to be induced under growth in low Mn2+ (0.1 μM) in the wild-type strain and also when the PsaR regulator is inactivated. Taken together, these findings suggest that Mn2+ acts with PsaR to cause the observed repression of this regulon. Mn2+ was the only variable in these experiments. Because Fe2+, Mg2+, Ca2+, and Zn2+ concentrations were nonlimiting and were kept constant in all our experiments, it is unlikely that these cations affected the PsaR-dependent regulation observed here. Inactivation of psaR was not judged as likely to create any gene expression change through a polar effect, since the gene immediately downstream of psaR is a transposase that is convergently transcribed with psaR.
PsaR affects transcription of multiple genes via expression of rlrA. Several of the genes under study, rrgA, rrgB, rrgC, and srtB, are clustered in the genome and located near the putative regulator, rlrA (Fig. 1B). Hava et al. demonstrated that RlrA activates the transcription of rrgA, rrgB, rrgC, and srtB (21). To confirm that this gene cluster is regulated by RlrA, we constructed an rlrA mutant strain of TIGR4. The expression of rrgA, rrgB, rrgC, and srtB in the wild-type TIGR4 strain and the rlrA mutant was examined. All four genes were expressed at almost undetectable levels in the rlrA mutant strain, confirming the results of Hava et al. (results not shown) (21). This raised the possibility that PsaR influences the expression of RlrA-regulated genes by repressing transcription of rlrA. When examined by slot blot analysis, the expression of rlrA was lower in the wild-type TIGR4 than in the psaR mutant under conditions of high Mn2+ (Fig. 2B and C). This indicated that PsaR affected transcription of rlrA, but the magnitude of the change in expression (twofold) was significantly less than in other PsaR-regulated genes. Thus, PsaR may be affecting the transcription of rrgA, rrgB, rrgC, and srtB through its effect on the expression of rlrA. Since RlrA is a transcriptional activator, a minimal increase in its expression can lead to a greater increase in the expression of its target genes. Thus, the relief of repression of the activator leads to an increase in the expression of rrgA, rrgB, rrgC, and srtB. The unlinked pcpA gene exhibited increased expression in the psaR mutant strain but was not affected in the rlrA mutant strain (data not shown), indicating that pcpA expression is not influenced by RlrA.
Inactivation of psaR and pcpA differentially impact carriage and pneumonia. Because PsaR represses the Mn2+ transporter psaABC and because Mn2+ levels are believed to be much higher in the nasopharynx than in the lung, we examined the effects of psaR mutations on virulence in murine models for pneumonia and nasopharyngeal carriage. Intranasal infection with strain EF3030 results in carriage and/or pneumonia depending on the infection protocol but does not progress to bacteremia and sepsis at the doses used in this study (11). For this purpose, psaR, pcpA, and psaR pcpA strains were constructed in the EF3030 background as described in Materials and Methods. EF3030 was chosen because the mouse pneumonia model is most like pneumonia in humans for this strain (11). The EF3030 strain lacks the rlrA-regulated gene cluster, so these results are relevant only to the other PsaR-regulated genes. Slot blot analysis confirmed that PsaR-mediated regulation of the psa operon and pcpA occurs in EF3030 (results not shown).
A reasonable prediction for the Mn2+-dependent repression was that the greatest impact of inactivation of psaR would be observed in the higher Mn2+ environment of the nasopharynx. This is because it is under high Mn2+ that regulated gene expression varies. To determine whether the mutant strains were deficient in the ability to establish nasopharyngeal carriage, we examined the colonization of each of the mutants compared with EF3030 following intranasal infection with 106 CFU. The bacteria were given in a 10-μl volume to nonanesthetized mice to produce carriage without pneumonia (11). In this infection model, no significant difference in nasopharyngeal colonization was seen between the mutants and the wild-type strains (Fig. 3). These results suggest that neither psaR nor pcpA is required for the establishment of carriage of pneumococci.
To determine the effects of the inactivation of psaR and pcpA in pneumonia, mice were inoculated with 107 CFU in a 40-μl volume while anesthetized to ensure aspiration of pneumococci. Each of the mutant strains showed attenuated virulence in the lung, with a significant decrease in the number of bacterial CFU/lung compared to that of EF3030 (Fig. 4) (P 0.0001 for each). The results for the pcpA mutant strain support a role for PcpA in bacterial persistence within the lung. There was clear attenuation in the virulence of the pcpA mutant strain. The attenuated growth of the psaR mutant strain in the lung is puzzling. If the lung were subject to lower Mn2+ conditions than the nasopharynx, then PsaR repression would be relieved, and there would be little phenotypic differences between the mutant and wild-type strains. The psaR mutant strain should be expressing the Psa transporter encoded by psaABC, as well as the surface protein PcpA at wild-type levels in EF3030 (Fig. 2C). The absence of any of these PsaR-regulated proteins, as judged by results with knockout mutants, attenuates bacterial growth in the lung (20, 21). It is possible that the mucosal surfaces in the lung contain some levels of Mn2+, allowing for some PsaR-mediated repression to occur (that is relieved in the mutant strain). Alternatively, it is possible that the attenuation of the psaR mutant strain in the lung may be a consequence of overexpression of one or more of the regulated gene products. The psaR pcpA double mutant was slightly more attenuated than either single mutant strain, suggesting that there are two separate phenotypic impacts in the single mutant strains.
Inactivation of psaR does not affect adherence. It has previously been hypothesized that the adherence defect observed in psaA mutants may be due to the disruption of the activity of an Mn2+-dependent transcriptional regulator (44). To test this hypothesis, the adherence of TIGR4 and its psaR mutant to a nasopharyngeal cell line was examined. The psaA mutant strain previously shown to have altered adherence in this assay (31) was included as a control. Binding curves were generated for each strain, and the psaR mutant was found to adhere to the D562 cells at wild-type levels throughout the curve, while the psaA mutant exhibited a reduction in adherence (Fig. 5). These data indicate that PsaR is not responsible for the regulation of the adherence function that is defective in psaA mutants, but this does not eliminate the possibility that Mn2+ may be involved in the regulation of adherence.
DISCUSSION
The control of Mn2+ homeostasis is expected to be important to the bacterial cell, and PsaR is important to this control in pneumococci. On the basis of our analysis of transcription patterns, PsaR appears to function as a repressor when sufficient Mn2+ is present. PsaR has a predicted metal binding site and is likely to bind to its targets when Mn2+ is present. PsaR shares 76% similarity and 65% amino acid sequence identity with ScaR, the Mn2+-dependent repressor of S. gordonii (30). The target sequence for ScaR binding has been identified, and the binding site for ScaR is 90% identical to a putative PsaR operator located in the promoter region of the psa operon (30). Additionally, the residues responsible for the selective binding of Mn2+ or Fe2+ by MntR or DtxR, respectively, have been previously identified (19), and the sequence of PsaR suggests it would bind Mn2+. This mode of action for PsaR was strongly supported by our data: inactivation of PsaR resulted in the derepression of a set of genes that are also derepressed when low Mn2+ concentrations are present in the wild-type strain.
PsaR-regulated genes were located in three different regions of the chromosome, indicating that PsaR is a multigene regulator. A model for PsaR regulation is presented in Fig. 6. In this model, the DNA-binding activity of PsaR is activated by the binding of Mn2+, which then represses the transcription of psaBCA, pcpA, and rlrA. Repression of rlrA leads to lower expression of rrgA, rrgBC, and srtBCD, since these genes are activated by RlrA. The potential regulatory cascade with PsaR and RlrA may explain the effect that psaR inactivation and Mn2+ concentration have on the expression of rrgA, rrgB, rrgC, and srtBCD. This regulatory cascade is evidenced by a greater change of expression in RlrA-regulated genes than in rlrA itself in comparing expression between psaR and TIGR4. In essence, the cascade allows the rlrA regulon to respond to the external Mn2+ concentration.
As an essential trace element, Mn2+ is extremely limited within the human body, where it may be complexed with carrier proteins (50). The highest concentration of in vivo Mn2+ appears to be in saliva, where it is 36 μM (12, 32). Thus, while pneumococci are in the nasopharynx, Mn2+ is presumably relatively abundant and expression of psaBCA, pcpA, and the rlrA-controlled genes would be repressed. When pneumococci invade the lung or bloodstream, the levels of Mn2+ are much lower (20 nM given for serum/plasma) (29, 50). Expression of the psa operon would be increased, inducing the high-affinity transporter in order to obtain Mn2+ that the bacterium needs to survive. Consistent with this model, transcript levels of psaA were increased in bacteria recovered from blood of an infected mouse compared to bacteria grown in vitro (43). Also, the repeated recovery of psaA, psaB, or psaC in a screen for in vivo-induced genes in a murine respiratory tract infection model is consistent with this scenario (36). The external change in Mn2+ concentration is clearly one factor that signals the bacterium to change its expression of virulence factors in response to its environment.
We identified additional genes outside of the psa transporter itself that are repressed by PsaR in response to Mn2+ concentrations. These include pcpA, rrgA, rrgB, rrgC, and srtB. PcpA is a choline-binding protein that contains leucine-rich repeats (49), which can often be involved in protein-protein interactions (33). On the basis of this, it was hypothesized that PcpA functions as an adhesin (49). Signature-tagged mutagenesis identified pcpA as being important for both pneumonia and bacteremia (20). In our study, the pcpA mutant was able to colonize the nasopharynx similar to the wild type, but Hava and Camilli found that a pcpA mutant was less effective in colonization when studied using a competitive carriage model (20). Our data supports a role for PcpA in the persistent establishment of growth during pneumonia, even in the absence of competition with the wild-type strain.
RrgA, RrgB, and RrgC are putative surface proteins that contain YPXTG, IPXTG, and VPXTG motifs, respectively, at their carboxy-terminal ends. These putative recognition sites are predicted to be recognized by sortase enzymes, which would then anchor these proteins to the cell wall. The rrgABC genes are clustered with the srtBCD operon on the chromosome (Fig. 1B). The proximity of the putative sortases to rrgA, rrgB, and rrgC and the unusual sorting motifs suggest that the sortases SrtB, SrtC, and/or SrtD specifically anchor these gene products. No specific binding activities for the Rrg proteins have been reported, although their weak homology to some microbial surface components recognizing adhesive matrix molecules, which often bind to fibronectin or fibrinogen, has suggested a possible adhesive function (9, 20). All of the genes in this cluster are positively controlled by rlrA (21). The rlrA-controlled region has an atypical GC content relative to the rest of the genome, and it is not common to all pneumococcal strains, indicating the possibility of horizontal transfer (28, 58). In the published studies, rrgA and srtD mutants showed reduced virulence in competitive pneumonia models of infection (20-22). Additionally, rrgC and srtC mutants exhibited an increased virulence (higher competitive index) for colonization in a carriage model, although rrgC did not reach significance (20). All of the rlrA islet genes are up-regulated in the TIGR4 psaR mutant strain. In the EF3030 psaR strain, psaABC and pcpA are similarly up-regulated. However, the genes in the rlrA islet are not present in this strain background.
Strain EF3030 is used in the mouse pneumonia model, because it is rapidly cleared from the blood, it causes pneumonia that is verifiable by histology, and there is an absence of bacteremia. This is similar to the natural disease state in humans (11). The net effect of both the pcpA mutation and the psaR mutation in this model was an attenuation of virulence. As EF3030 lacks the rlrA cluster, in this strain the major impact of the psaR mutation is likely to be felt through the elevated expression of either PcpA or the Psa transporter. Alternatively, it is possible that additional genes not found in our in vitro system may be regulated by psaR in the in vivo setting, contributing to the phenotype.
The expression of pcpA was also regulated in response to Mn2+ in our work, leading to a predicted lower level of expression in the nasopharynx and a higher level of expression in the lung and blood. Because PcpA did not appear to be required for colonization of the nasopharynx in our carriage model (Fig. 3, pcpA mutant), we predict that it may play its major role in the lung. This would be consistent with its regulation, since pcpA is repressed by PsaR under the Mn2+ conditions similar to those in the nasopharynx. The unregulated expression of PsaR-regulated proteins also did not reduce colonization, judged by the similar CFU loads in the wild-type and psaR mutant strains. Together, these observations suggest that the major impact of regulation in response to Mn2+ is felt in the pneumonia model. Our data imply that both too little surface protein in the pcpA mutant and too much surface protein in the psaR mutant may be detrimental.
These results highlight the complexity involved in understanding the nature of the phenotype of any dysregulation. Strain EF3030 lacked the rlrA cluster, but in another pneumococcal strain, the impact of a psaR mutation or the Mn2+ concentration may be felt through the rlrA cluster. Hemsley et al. have found that the MgrA regulator acts as a repressor on RlrA, but mgrA mutations also result in the attenuation of virulence in pneumonia (24). Results suggested that although some rlrA-regulated products may be required for full virulence in pneumonia, the elevated levels of one or more of these gene products that occurred under the loss of negative control by mgrA was also detrimental to the development of full virulence. A similar scenario may occur for PsaR regulation, either through PcpA or through the rlrA cluster in strains containing it.
The psaBCA operon has been shown to be activated by a two-component system, RR04/HK04, in strain TIGR4 but this did not occur in strain D39 and 0011389 (39). RR04 appeared to interact separately with the psa operon, rather than indirectly through psaR, based on the observation that psaR transcription was unaltered in the RR04 mutant (39) and that other PsaR-regulated genes, pcpA or the rlrA regulon, were not similarly affected. This regulatory system may put the expression of the psa operon under the control of an environmental signal in addition to Mn2+; but this appears to be specific to TIGR4.
This is the first demonstration of a pathogen using Mn2+ as a signal to modulate the expression of virulence genes which may have different impacts in different host sites. The disruption of this regulation negatively impacts virulence in a pneumonia model. We have also demonstrated that PcpA, a PsaR-regulated gene, has a role in the persistence of pneumococci in pneumonia but does not have a role in colonization of the nasopharynx. The control of expression of additional PsaR-regulated genes involves multiple factors, and more work is necessary to determine how these regulators interact to control the expression of multiple pneumococcal virulence factors.
ACKNOWLEDGMENTS
These studies were conducted with the support from NIH grants AI053749, AI40645, and AI21548. Jason Johnston was supported by NIAID training grant T32 AI 07041.
We thank Martina Ochs for her input in reviewing the manuscript prior to publication. We thank Yvette Hale for her assistance with the virulence studies. Additionally, we thank Priya Balachandran for help with the RNA procedures.
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