Involvement of potD in Streptococcus pneumoniae Polyamine Transport and Pathogenesis
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感染与免疫杂志 2006年第1期
Department of Microbiology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, Mississippi 39216
Research Service, Veterans Affairs Medical Center, 1500 Woodrow Wilson Drive, Jackson, Mississippi 39216
Department of Chemistry, University of Missouri—Rolla, 342 Schrenk Hall, Rolla, Missouri 65409
ABSTRACT
Polyamines such as putrescine, spermidine, and cadaverine are small, polycationic molecules that are required for optimal growth in all cells. The intracellular concentrations of these molecules are maintained by de novo synthesis and transport pathways. The human pathogen Streptococcus pneumoniae possesses a putative polyamine transporter (pot) operon that consists of the four pot-specific genes potABCD. The studies presented here examined the involvement of potD in polyamine transport and in pneumococcal pathogenesis. A potD-deficient mutant was created in the mouse-virulent serotype 3 strain WU2 by insertion duplication mutagenesis. The growth of the WU2potD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2potD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (-dimethyl-fluroornitithine) and MGBG [methylgloxal-bis (guanyl hydrazone)]. The mutant strain also showed a significant attenuation in virulence within murine models of systemic and pulmonary infection regardless of the inoculation route or location. These data suggest that potD is involved in pneumococcal polyamine transport and is important for pathogenesis within various infection models.
INTRODUCTION
Polyamines such as putrescine, spermidine, and cadaverine comprise a group of ubiquitous aliphatic, polycationic molecules which are present in all cells (21, 33, 44, 45). These molecules are necessary for normal cell growth and have been associated with a wide variety of physiological processes, primarily through their interaction with ATP and polyacids such as DNA and RNA (19). Polyamines have been shown to exert effects on virtually all aspects of cellular physiology involving nucleic acids, including DNA synthesis, transcription, and translation (12a). Intracellular polyamines are derived from both de novo synthesis from amino acids and intracellular uptake from the environment (20, 53). The intracellular levels of polyamines are tightly regulated by multiple mechanisms involving both biosynthesis and transport processes. Polyamine synthesis is regulated in part by antizyme degradation of ornithine decarboxylase, a key enzyme which decarboxylates ornithine to form putrescine (13, 41) as well as feedback inhibition by the amino acid ornithine or excess levels of polyamines (14, 45). In Escherichia coli, transcription of genes encoding a polyamine transporter is inhibited by intracellular putrescine and by excess cellular concentrations of gene products (1, 26).
Although polyamines have been shown to have multiple effects on protein synthesis and cell proliferation for all cell types, polyamine uptake and synthesis in pathogenic bacteria has not been well studied. In E. coli, a four-gene operon encodes an ABC transporter for putrescine and spermidine and a separate four-gene operon encodes a putrescine-specific transporter (21). E. coli also expresses a transmembrane protein which functions as a putrescine-ornithine antiporter (27). E. coli mutants unable to synthesize polyamines de novo grow at a much slower rate than wild-type cells (46), as do mutants which cannot transport exogenous polyamines from the immediate environment (25). Together, these findings suggested that E. coli depends upon both transport and synthesis processes to maintain optimal intracellular polyamine levels.
Most of the known details about the physiology of polyamines in prokaryotes have been derived from E. coli studies, and knowledge of polyamine metabolism and transport in other human pathogens consists only of functional genomic analysis of the completed genome projects. Many clinically relative bacterial species contain genes associated with a superfamily of amino acid-polyamine-organocation transporters as determined on the basis of sequence analysis (22), but almost nothing is known about their function during in vitro growth or in a human host. Shigella flexneri mutants unable to synthesize modified nucleosides necessary for tRNA synthesis can utilize supplemental putrescine to restore virulence gene expression (10). Enterococcus faecalis can use agmatine as another energy source and takes up this polyamine by an agmatine-putrescine antiporter (8). Putrescine and cadaverine have been found in the peptidoglycan in Veillonella and Selenomonas spp. (24).
The human pathogen Streptococcus pneumoniae (pneumococcus) is the most common bacterial cause of pneumonia, and S. pneumoniae infection can also lead to septicemia and meningitis (30). While the polysaccharide capsule of pneumococci is considered to be one of the most important virulence factors of this organism, many surface-associated and secreted proteins are now being studied for their role in pathogenesis and protective immunity (5, 23, 42). Pneumococcal genes with homology to a polyamine transporter (Pot) operon in E. coli have been implicated in the pathogenesis of pneumococcal infection in a mouse model of septicemia and pneumonia (34, 51). The sequenced genomes of pneumococcal strains TIGR4 and R6 have been published, and both strains contain the four contiguous genes potABCD that encode a putative spermidine-putrescine transporter (17, 47). Through functional genomic analysis using the National Center for Biotechnology Information BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), the function of each pot gene product was determined. The potA gene contains consensus Walker A and Walker B sites which define an ATP-binding cassette (39); PotA is therefore predicted to be a typical ATP-binding protein. The proteins encoded by potBC contain multiple -helical hydrophobic domains, a typical feature of integral membrane proteins, and are predicted to form a polyamine-specific channel through the pneumococcal cell membrane. The potD gene encodes a surface-associated spermidine- and putrescine-binding protein that has high homology to PotD in E. coli. Pneumococcal PotD contains a typical gram-positive signal peptide but does not possess motifs characteristic of lipoproteins, sortase-processed proteins, or choline-binding proteins. Figure 1 diagrams the functional genomics of the potABCD operon within Streptococcus pneumoniae. Because of the surface location and polyamine-binding function of PotD, this protein is potentially associated with pneumococcal virulence. To date, neither polyamine transport nor biosynthesis within the pneumococcus has ever been studied. In this paper, we define the actual involvement of the potD gene in polyamine transport and verify its importance in pneumococcal pathogenesis. A potD deletion mutant was created in the encapsulated, mouse-virulent serotype 3 strain WU2. Phenotype differences associated with potD inactivation within the WU2potD mutant were investigated within in vitro and in vivo growth models. The actual involvement of the potD gene in pneumococcal polyamine transport was verified by comparing the in vitro growth kinetics of WU2potD and wild-type WU2 in the presence of polyamine biosynthesis inhibitors. The importance of potD in pneumococcal pathogenesis was demonstrated in murine models of systemic and pulmonary infection. By comparing the in vitro and in vivo phenotypes of WU2potD, the involvement of polyamine transport in pneumococcal virulence was conclusively shown. This is the first time that polyamine transport has been associated with the ability of a human pathogen to cause disease.
MATERIALS AND METHODS
Bacterial strains, DNA, and growth conditions. All bacterial strains, plasmids, and PCR primers used for this work are listed in Table 1. Streptococcus pneumoniae strain WU2 expresses a serotype 3 capsule (4) and was used in all experiments. This strain is highly virulent in a mouse model of sepsis (3). To create frozen stocks of bacteria, cells were grown to early and mid-exponential phase in the rich medium Todd-Hewitt yeast extract (THY) broth at 36°C in 5% CO2 and the cells were collected by centrifugation. The cells were then washed twice in sterile phosphate-buffered saline (PBS; pH 7.0) at 4°C and stored at –80°C in PBS with 20% glycerol for bacterial stock cultures. Bacterial stocks were quantitated by counting CFU in serial dilutions plated on THY plates containing 4% sheep erythrocytes (Colorado Serum Co., Denver, CO). Bacteria from frozen stocks were used to inoculate THY or a chemically defined medium (CDM) (JRH Bioscience, Lenexa, KS) which has been previously described (50). The CDM was supplemented with filter-sterilized choline chloride, ethanolamine, spermidine, or putrescine to concentrations listed for specific experiments. For inhibition experiments, THY or CDM containing 1 mg/ml of choline was supplemented with 100 μM DFMO (-dimethyl-fluroornitithine), an inhibitor of ornithine decarboxylase (Sigma, St. Louis, MO) and 100 μM MGBG [methylgloxal-bis (guanyl hydrazone)], an inhibitor of adenosylmethionine decarboxylase (Sigma, St. Louis, MO) (2, 50, 53). For in vivo experiments, frozen stocks of WU2 and WU2potD were used to inoculate THY and the cells were grown to the exponential phase before collection by centrifugation. All cultures were grown in disposable polystyrene tubes and incubated in 5% CO2 at 36°C. Bacterial growth in liquid media was monitored by measuring the absorbance of resuspended cells at 600 nm in 1 ml cuvettes with a spectrophotometer at various time intervals during growth. All cultures were vortexed in the presence of glass beads (100 microns) (Sigma, St. Louis, MO) for two 30-s intervals to physically separate the pneumococcal cells that were growing as long viscous threads that frequently settled to the bottom of culture tubes. Desolution of the long chains was validated by gram staining before and after vortexing in the presence of glass beads. Terminal subcultures were done after all experiments by plating bacteria from the liquid media onto blood agar plates and testing for optochin sensitivity to assure purity and identity of the cultures. Bacteria concentrations (approximately 1 x 106 to 1 x 108) were estimated by spectrophotometer (A600) based on previously performed standard curves plotting A600 values against the number of viable cells for WU2 and further confirmed by viable CFU counts on sheep blood agar plates.
All DNA templates were prepared with genomic DNA isolated by collecting the bacterial cell pellet from a culture in exponential-phase growth by centrifugation and extracting the DNA as previously described (38). Growth medium and culture methods for genetic transformation were performed as previously shown (29, 35). Antibiotic concentrations in THY or blood agar plates were 100 μg/ml erythromycin for pneumococcal transformant selection or 100 μg/ml apramycin for propagation of E. coli containing pCZA342potD.
PCR amplification. All products below 5 kb were amplified using a Taq SuperPak DNA polymerase system (Sigma, St. Louis, MO), and all products above 5kb were amplified using an AccuTaq LA DNA polymerase system (Sigma, St. Louis, MO); for all amplification processes, the cycling parameters recommended by the manufacturer were used. All primers used are listed in Table 1.
Transformation and construction of mutant strain. All plasmids, primers, and S. pneumoniae strains utilized for this work are described in Table 1. To construct potD disruption vectors, an internal portion of the potD gene was amplified by PCR with intpotD1 and intpotD2 primers and ligated into suicide vector pCZA342 (Table 1). Plasmid pCZA342 (18) encodes apramycin (for selection in E. coli) and erythromycin (for selection in S. pneumoniae) resistance markers. Correct plasmid constructs (pCZA342potD) were confirmed by restriction digestion using EcoRI (Promega, Madison, WI) and PCR amplification with primers specific for the internal region of potD (intpotD1 and intpotD2 in Table 1). A S. pneumoniae RX1potD strain containing a disrupted potD gene was constructed by insertion-duplication mutagenesis with pCZA342potD according to previously described transformation protocols utilizing competence-stimulating peptide 1 (29). The RX1potD construct was confirmed by PCR with AccuTaq LA DNA polymerase (Sigma, St. Louis, MO) by use of FLpotD1 and FLpotD2 primers (Table 1). Competent WU2 cells were then transformed with chromosomal DNA from RX1potD and competence-stimulating peptide 2 as previously performed (35). The transformants were selected on 4% sheep blood agar plates supplemented with erythromycin (100 μg/ml). Each erythromycin-resistant transformant was backcrossed three times with wild-type WU2 background as previously described (28, 36). The WU2potD construct was confirmed by PCR with AccuTaq LA DNA polymerase (Sigma, St. Louis, MO) by use of FLpotD1 and FLpotD2 primers (Table 1) and by Southern blotting. Amplification of the potC gene was used as an internal control to verify that only the potD gene was disrupted by the transformation procedure in WU2potD. All mutants were stable after three 12-hour growth cycles in THY without antibiotic selection, with 100% of the 25 colonies tested retaining erythromycin resistance.
Southern blot analysis and preparation of hybridization probes. Phenol-chloroform-extracted DNA from strains WU2 and WU2potD was digested for 6 h with XbaI (Promega, Madison, WI) at 37°C according to the manufacturer's instructions. The digested DNA was subjected to electrophoresis in a 1% (wt/vol) agarose gel in Tris-borate-EDTA buffer for 15 h at a constant 24 V. Before blotting, the gel was immersed and gently agitated at room temperature in 250 mM HCl (10 min), 1.5 M NaCl-0.5 M NaOH (30 min), and 1.5 M NaCl-0.5 M Tris-HCl (pH 7.5) (30 min), with water rinses between treatments. DNA was transferred from the agarose gel to a Hybond-N+ (Amersham, Arlington Heights, Ill.) membrane by capillary blotting and was cross-linked to the membrane by using a UV cross-linker (Stratalinker; Stratagene, La Jolla, Calif.). The membrane was prehybridized with Dig EasyHyb buffer (Roche Applied Science, Indianapolis, IN) overnight and hybridized for 12 h with a digoxigenin-labeled probe made from a PCR-amplified internal region of the potD gene amplified with intpotD1 and intpotD2 primers. The PCR-amplified internal potD probe was prepared with a Dig High Prime DNA labeling system (Roche Applied Science, Indianapolis, IN). After hybridization with the probe, the membrane was washed twice in 1x SSC (SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% (wt/vol) sodium dodecyl sulfate at room temperature with gentle agitation for approximately 5 min per wash. The membrane was then washed twice with prewarmed (55°C) 0.1x SSC-1% (wt/vol) sodium dodecyl sulfate at 55°C for 15 min with gentle agitation. Visualization of the Southern blot was performed with a digoxigenin luminescent detection system (Roche Applied Science, Indianapolis, IN) following the manufacturer's instructions. Each membrane was exposed to X-ray film in a cassette for 15 min in order to visualize the location of the hybridized probe. The molecular weights of the hybridization signals were determined by comparison with a standard molecular weight ladder and a known full-length potD PCR fragment.
In vivo mouse studies. Experiments were all performed using CBA/CaHN-Btkxid (CBA/N) mice (Jackson Laboratory, Bar Harbor, Maine) and bred within the animal care facility (Veterans Affairs Medical Center, Jackson, MS). CBA/N mice have an X-linked inability to produce normal humoral antibody responses to a group of thymus-independent antigens, including type 3 pneumococcal capsular polysaccharide, making this mouse strain more susceptible to pneumococcal infections (4). A total of 1x 104 CFU/ml of either strain WU2 or WU2potD cells in PBS were injected either intraperitoneally (i.p.) or intravenously (i.v.) in a volume of 0.1 ml into 3- to 5-month-old mice for a model of systemic infection as previously described (6). For a pulmonary model of infection, pneumococci were introduced into the lungs of 3- to 5-month-old CBA/N mice by direct intratracheal (i.t.) inoculation. The animals were anesthetized by being placed for 30 s into an air-tight bell jar that contained a sterile gauze pledget moistened in 3 ml of Isoflurane (Abbot Laboratories, Abbott Park, Illinois), which was placed in the bottom of the bell jar, beneath an elevated platform preventing direct contact between the mouse and the anesthetic. After anesthesia was established, the mice were inoculated with approximately 1 x 104 CFU of WU2 or WU2potD cells. Mice were held in vertical suspension for 5 min after inoculation to facilitate deep penetration of the inoculums. For mouse survival curves, mice infected with 104 CFU of either WU2 or WU2potD cells were observed for extended time intervals and the overall survival of each mouse was recorded.
Polyamine biosynthesis inhibition assays. Wild-type WU2 and WU2potD cells were grown in separate 10-ml cultures to mid-exponential phase (optical density at 600 nm [OD600] of 0.5 to 0.6) in THY under the previously stated conditions. The cultures were collected by centrifugation, washed three times in a glucose citrate buffer (100 mM sodium citrate [pH 5.5], 2% D-glucose) to ensure an efficient removal of any contaminating exogenous polyamines, and resuspended in sterile PBS buffer (pH 7.0) to a final concentration of 106 CFU/ml of suspension. A total of 105 CFU of washed WU2 or WU2potD cells were used to inoculate 25 ml of THY or CDM supplemented with 1 mg/ml of exogenous choline and one of the following combinations: 100 μM MGBG and DFMO; 100 μM MGBG and DFMO and 50 μg/ml putrescine; or 100 μM MGBG and DFMO and 50 μg/ml spermidine. Culture growth was monitored every 2 h for 12 h by measuring culture turbidity with a spectrophotometer at OD600.
Intracellular polyamine concentration determination. Putrescine, cadaverine, and spermidine concentrations were determined by high-performance liquid chromatography as described previously (9) with some modifications. Wild-type WU2 and WU2potD cells were grown in separate 40-ml cultures to mid-exponential phase (OD600, 0.5 to 0.6) in THY or CDM plus choline with or without 100 μM MGBG and DFMO. The cultures were collected by centrifugation and washed three times in a glucose citrate buffer (100 mM sodium citrate [pH 5.5], 2% D-glucose) and twice in sterile PBS buffer (pH 7.0). The cells were resuspended in 5% HClO4, vigorously vortexed, and allowed to chill at 4°C for 12 h. After centrifugation, the supernatant was removed to a new tube. The bacterial cell extract solution was neutralized with 8.0 M NaOH to a pH of between 3 and 4 in order to precipitate the proteins. The supernatant was removed after centrifugation at 10,000 x g for 15 min at 4°C. The precipitates were washed two times with 0.1 M HClO4, and all of the supernatants were combined into one tube. The polyamines were derivatized with fluorescein-5-isothiocyanate and quantified by the capillary electrophoresis method as described previously (9).
Statistical analysis. All growth kinetics data represent experiments performed two to three times and are presented as the mean values for all samples. Error bars represent the standard error of the mean for each corresponding data set. Survival curve data were compared using Kaplan-Meier survival curves, and all statistical significance was determined using log rank tests. The MedCalc statistical program was used for all statistical analysis.
RESULTS
Generation of a potD mutant by gene disruption insertion mutagenesis. The gene for potD was disrupted by insertion duplication mutagenesis (Fig. 2A), using pCZA342 as the suicide vector that contained a cloned internal portion of the potD gene for gene targeting and an erythromycin resistance cassette for mutant isolation. The internal portion of the potD gene was obtained using PCR amplification of a 550-bp internal fragment of the potD gene, D39 chromosomal DNA, and primers intpotD1 and intpotD2 (Table 1). Initially, the potD gene was disrupted in the unencapsulated laboratory strain RX1 (data not shown). The potD-knockout mutants were isolated after transformation based on transformant resistance to 100 μg/ml of erythromycin. The resulting mutated chromosomal DNA preparations from RX1potD were used to create a potD-knockout mutant in the highly mouse-virulent capsular serotype 3 WU2 strain. Each mutant acquired from the transformation procedure was identified by resistance to 100 μg/ml erythromycin and backcrossed three times with wild-type WU2 chromosomal DNA as described in Material and Methods. The WU2potD construct was confirmed by PCR using primers specific for the full-length potD gene (FLpotD1 and FLpotD2 in Table 1; Fig. 2B) and by Southern blotting (Fig. 2C). For PCR confirmation, potD amplified from WU2potD was expected to be 7.5 kb while potD from wild-type WU2 was expected to be 1.1 kb (Fig. 2B). For the Southern blot, the expected hybridization position was 29,563 kb for WU2potD and 23,063 kb for wild-type WU2 (Fig. 2C). Both PCR and Southern blotting confirmed that WU2potD contained a plasmid insertion mutation within potD that inactivated the gene.
In vitro growth studies of WU2potD in rich medium. Growth of strain WU2potD was compared to that of the parent WU2 strain in the undefined complete medium THY and in a completely defined medium that does not contain exogenous polyamines. Growth of WU2potD was identical to that of wild-type WU2 in both THY (Fig. 3A) and in CDM-50 μg/ml choline (Fig. 3B). As expected, the growth kinetics of WU2potD in vitro were identical to those of wild-type WU2. The published pneumococcal genomes contain various enzymes predicted to be involved with the biosynthesis of polyamines from amino acids (Table 2). Because polyamine concentrations are regulated by both synthesis and transport mechanisms, the transport-deficient WU2potD strain could still maintain adequate intracellular concentrations of polyamines due to de novo synthesis of endogenous polyamines.
Effects of polyamine synthesis inhibition on WU2potD in vitro growth. Polyamine content within cells is regulated by both polyamine transport and biosynthesis. Even with potD inactivation, WU2potD would still be able to concentrate intracellular polyamines through putative de novo synthesis pathways. In order to determine how both potD inactivation and de novo polyamine synthesis inhibition would effect the growth of WU2potD, the two polyamine synthesis inhibitors MGBG and DFMO were added to either THY or CDM containing 1 mg/ml choline. For the experiment, 105 CFU of either wild-type WU2 or WU2potD were grown in THY alone or in the presence of both MGBG and DFMO (Fig. 4A). WU2 grown in the presence or absence of the inhibitors reached exponential growth at 2 h postinoculation. WU2potD grew identically to wild-type WU2 in the absence of the inhibitors. However, WU2potD grown in THY plus inhibitors had delayed growth, reaching exponential growth at 7 h postinoculation. An initial inoculum of 105 CFU of either WU2 or WU2potD was also cultured in CDM-1 mg/ml choline with or without the addition of both MGBG and DFMO (Fig. 4B). WU2 reached exponential-phase growth at 10 h postinoculation, regardless of whether the inhibitors were present or not (Fig. 4B). WU2potD grown without the addition of the inhibitors also reached exponential growth at 10 h postinoculation. Only WU2potD growth in the presence of the inhibitors MGBG and DFMO was affected (Fig. 4). WU2potD growth in medium containing both DFMO and MGBG was greatly delayed; the cells reached exponential-phase growth 16 h postinoculation. WU2 growth was not affected by the addition of MGBG and DFMO to the culture medium. WU2potD growth, however, was delayed but not prevented by the addition of MGBG and DFMO to the culture medium, indicating that the inhibition of polyamine synthesis was most important within WU2potD during the initial growth steps.
Polyamines have been shown to be involved in various steps of cell growth. Both WU2 and WU2potD were able to grow in the presence of MGBG and DFMO, although WU2potD grew much more slowly, possessing an extended lag phase compared with WU2 results. To determine whether the final intracellular polyamine concentrations for both WU2potD and WU2 were similar, the concentration of intracellular polyamines was compared for both WU2 and WU2potD. In Table 3, the intracellular concentrations of the polyamines cadaverine, spermidine, and putrescine are compared for WU2 and WU2potD grown in THY or CDM plus 1 mg/ml choline with or without MGBG and DFMO added to the medium. The intracellular levels of the all three polyamines for WU2potD grown in both THY and CDM plus 1 mg/ml choline were similar, and these concentrations did not differ much after the addition of MGBG and DFMO (Table 3). For WU2, the intracellular levels of spermidine and putrescine did not differ much for both THY and CDM plus 1 mg/ml choline with or without MGBG and DFMO (Table 3). Interestingly, the addition of MGBG and DFMO to the both mediums did increase the intracellular concentrations of cadaverine within WU2 fivefold for cells grown in THY and threefold for cells grown in CDM plus choline (Table 3).
Alternative polyamine transport systems have been previously reported for E. coli. The unaffected growth kinetics along with the increased levels of intracellular cadaverine within WU2 in the presence of MGBG and DMFO supported the idea of the existence of alternative polyamine transport systems in the pneumococcus as well. To determine whether alternative polyamine transport systems for the polyamines putrescine and spermidine could exist in WU2potD, the mutant and WU2 were grown in CDM supplemented with 1 mg/ml of choline and 50 μg/ml exogenous putrescine or spermidine with or without 100 μM of DMFO and MGBG (Fig. ). The addition of 50 μg/ml exogenous putrescine or spermidine enabled WU2potD (Fig. 5B) to display growth kinetics identical to those of wild-type WU2 (Fig. 5A), even in the presence of both MGBG and DFMO.
The decreased proliferation rate of strain WU2potD but not strain WU2 resulting from the addition of DFMO and MGBG confirmed that PotD was involved in polyamine transport. Although WU2potD did display an extended lag phase compared with wild-type WU2 in the presence of MGBG and DFMO, both strains possessed comparable concentrations of intracellular polyamines once exponential-phase growth was reached. Also, the addition of exogenous putrescine or spermidine reinstated wild-type-like growth in WU2potD in the presence of MGBG and DFMO. These findings support the existence of potD alternative polyamine transport system(s) and possible alternative de novo biosynthesis pathways within the pneumococcus.
In vivo experiments in murine models of systemic and pulmonary infection. The potD gene was previously predicted to be associated with pneumococcal pathogenesis in mouse models of septicemia and pneumonia as determined by use of signature-tagged mutagenesis (34). To verify the involvement of potD in pathogenesis, we investigated the virulence of WU2potD within systemic and pulmonary infections. For systemic infection, mice were given i.p. (Fig. 6A) or i.v. (Fig. 6B) inoculations of 104 CFU of either wild-type WU2 or WU2potD. Five mice per bacterial strain were inoculated i.p. and 10 mice per bacterial strain were inoculated i.v. with either WU2 or WU2potD. For the pneumonia model, 10 mice were infected through an i.t. route with 104 CFU of either wild-type WU2 or WU2potD (Fig. 6C). For all infection models, a statistically significant delay in time existed before the occurrence of a terminal infection in the mice inoculated with WU2potD compared to the results seen with the mice infected with wild-type WU2. The P values for all infection models were as follows: for i.p. infection, 0.0116; for i.v., 0.0122; and for i.t., 0.0052. All murine infection models indicated that WU2potD had attenuated virulence regardless of the infection route or model tested.
DISCUSSION
Previously, a possible association of the putative polyamine transport gene potD with pneumococcal pathogenesis was suggested (34). In this study, we determined the in vitro and in vivo effects associated with the creation of the potD-deficient mutant WU2potD. Because WU2potD does not produce functional PotD, the mutant strain was unable to uptake polyamines through the activities of the transport system encoded by potABCD. Therefore, WU2potD offered the opportunity to determine whether potD was involved in polyamine transport and whether it is important in pneumococcal virulence. WU2potD displayed growth kinetics identical to those of wild-type WU2 in various rich mediums, indicating that the pot operon is not necessary for in vitro growth of the pneumococcus within such environments.
Although WU2potD had in vitro growth kinetics identical to those of wild-type WU2, the in vivo growth and associated virulence of the mutant strain were vastly affected by potD inactivation. WU2potD displayed significant virulence attenuation in murine models of both septicemia and pneumonia, and polyamine transport has been well established as an integral part of cell growth and proliferation. When a pathogen invades a potential host, the pathogen must adapt quickly to a new environment to multiply and evade the host immune system. Consequently, the pathogen would have to simulate the cellular processes of transcription and translation, both of which are processes in which polyamines would be actively involved. Therefore, polyamine transport would be important for the initial steps in pneumococcal infection. Because PotD is the polyamine-binding protein of the PotABCD system, inactivation of the associated potD gene would prevent polyamine uptake via the PotABCD system and decrease the amount of available polyamines within the bacterial cell as a whole. Although most cells obtain polyamines through multiple de novo synthesis pathways and transport systems, the removal of one transport element could significantly decrease the pool of available polyamines; during a time of environmental stress such as in vivo infection, this decrease of intracellular polyamines could affect the overall proliferation and virulence of the bacteria. The inactivation of the potD gene in the pneumococcus greatly decreased the pathogenesis of the bacteria within the host, regardless of the inoculation route or whether the infection was systemic or pulmonary in nature.
Because strain WU2potD displayed in vitro growth kinetics identical to those of the wild-type WU2 strain, the involvement of PotD in polyamine uptake needed to be defined. The polyamine de novo synthesis pathways of Streptococcus pneumoniae were inhibited to determine whether the inactivation of potD affected cell proliferation in a polyamine-deficient medium. The polyamine biosynthesis inhibitors DFMO and MGBG were utilized to stop putrescine production from ornithine and spermidine synthesis. WU2potD growth delay due to the addition of the polyamine biosynthesis inhibitors directly shows the involvement of the potD gene with polyamine uptake. Interestingly, WU2potD was able to grow in medium supplemented with DFMO and MGBG only (no exogenous polyamines were added), although the growth rate was much slower than the growth rate for wild-type WU2. These data suggested that WU2potD possibly possessed alternative means of polyamine obtainment. To determine whether WU2potD was able to obtain polyamines through pathways other than PotD-associated ones, the levels of intracellular polyamines for WU2 and WU2potD were compared for both strains during exponential-phase growth. Intracellular polyamine levels for both WU2 and WU2potD were similar for putrescine, spermidine, and cadaverine, as both strains entered exponential-phase growth in THY and CDM plus 1 mg/ml choline. The addition of MGBG and DFMO to THY and CDM plus 1 mg/ml choline did not alter the exponential-phase growth concentrations of putrescine, spermidine, or cadaverine in WU2potD or the concentrations of putrescine and spermidine in WU2. However, for WU2, the intracellular concentrations of cadaverine were greatly increased by the addition of MGBG and DFMO to the growth medium. These observations suggest that alternative polyamine biosynthesis and/or transport pathways may be present within the pneumococcus. Most bacterial cells have been show to possess a secondary pathway for putrescine production that is based on the conversion of the amino acid arginine into putrescine by arginine decarboxylase (ADC) (15, 31). Streptococcus pneumoniae may contain an inducible form of ADC that could facilitate pneumococci growth in the presence of DFMO and MGBG. Inducible ADC has been shown to exist in E. coli (46). Also, the increased concentrations of cadaverine within WU2 but not WU2potD after the addition of MGBG and DFMO to both THY and CDM plus choline indicates that potD could potentially be involved in cadaverine transport. Streptococcus pneumoniae may also upregulate the transport or biosynthesis of cadaverine in response to polyamine biosynthesis inhibition. Both published pneumococcal genomes contain cad, the gene that encodes lysine decarboxylase, an enzyme which converts the amino acid L-lysine into the polyamine cadaverine. In E. coli, cells in which the biosynthesis of putrescine is blocked by mutations upregulate the activity of the lysine decarboxylase and increase the production of cadaverine, a polyamine involved in E. coli protection from various environmental stresses (12, 14, 37). The complete reinstatement of wild-type growth in WU2potD by the addition of exogenous putrescine or spermidine was also surprising. The ability of WU2potD to use exogenous polyamines suggests that another polyamine transport system may exist within Streptococcus pneumoniae. Although different polyamines have been shown to share the same transport system(s), most cells have multiple polyamine uptake systems to ensure adequate intracellular concentration of polyamines (21). Although the published pneumococcal genomes do not seem to contain polyamine-specific transport genes other than potABCD, transport systems with multiple substrate specificities have been found in other cells. The uptake of putrescine and spermidine by Saccharomyces cerevisiae occurs via the activities of Gap1p, a protein previously believed to be mainly involved in the uptake of various amino acids (48). E. coli also encodes a putrescine-ornithine antiporter which has been shown not only to export putrescine during times of excess polyamines or acidic environmental pH but also to catalyze putrescine uptake (27). In both published pneumococcal genomes, YfnA shows 81.1% alignment to PotE in E. coli with a bit score of 112 out of 281, indicating potential similarities between protein functions (32). This gene could possibly be involved in polyamine antiporter activities within pneumococci.
Streptococcus pneumoniae is known to possess a multitude of virulence factors, from its capsule to various individual proteins that are necessary for an effective infection. The expression of virulence-associated genes has been shown to be unregulated upon in vivo infection (34). This upregulation of virulence gene expression results from the summation of different environmental influences and involves both the transcription of mRNA and the translation of various proteins. The concentrations of intracellular polyamines have been shown to influence the efficiency and fidelity of translation and to stabilize DNA, RNA, and various enzymes involved in both transcription and translation as well as to modulate the synthesis of various proteins at the translational level (31). The polyamine putrescine has been shown to restore virulence gene expression in the pathogen Shigella flexneri (10), to influence the formation of filaments in Candida albicans that are necessary for virulence in mouse models (16), and to be involved in host cell adherence by Trichomonas vaginalis (11). The data presented here show that the polyamine transport system encoded by potABCD influences the overall pathogenesis of Streptococcus pneumoniae within the murine host. The virulence attenuation occurs within multiple infection models and inoculation routes, supporting the importance of the potD gene and polyamine transport during the initial stages of infection. Because polyamines appear to have multiple effects within the pneumococcus, the study of polyamine function and uptake within this human pathogen is important and warrants further study. Also, the existence of many polyamine synthesis and transport inhibitors offers endless possibilities for new approaches to preventing human infection with Streptococcus pneumoniae.
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Research Service, Veterans Affairs Medical Center, 1500 Woodrow Wilson Drive, Jackson, Mississippi 39216
Department of Chemistry, University of Missouri—Rolla, 342 Schrenk Hall, Rolla, Missouri 65409
ABSTRACT
Polyamines such as putrescine, spermidine, and cadaverine are small, polycationic molecules that are required for optimal growth in all cells. The intracellular concentrations of these molecules are maintained by de novo synthesis and transport pathways. The human pathogen Streptococcus pneumoniae possesses a putative polyamine transporter (pot) operon that consists of the four pot-specific genes potABCD. The studies presented here examined the involvement of potD in polyamine transport and in pneumococcal pathogenesis. A potD-deficient mutant was created in the mouse-virulent serotype 3 strain WU2 by insertion duplication mutagenesis. The growth of the WU2potD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2potD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (-dimethyl-fluroornitithine) and MGBG [methylgloxal-bis (guanyl hydrazone)]. The mutant strain also showed a significant attenuation in virulence within murine models of systemic and pulmonary infection regardless of the inoculation route or location. These data suggest that potD is involved in pneumococcal polyamine transport and is important for pathogenesis within various infection models.
INTRODUCTION
Polyamines such as putrescine, spermidine, and cadaverine comprise a group of ubiquitous aliphatic, polycationic molecules which are present in all cells (21, 33, 44, 45). These molecules are necessary for normal cell growth and have been associated with a wide variety of physiological processes, primarily through their interaction with ATP and polyacids such as DNA and RNA (19). Polyamines have been shown to exert effects on virtually all aspects of cellular physiology involving nucleic acids, including DNA synthesis, transcription, and translation (12a). Intracellular polyamines are derived from both de novo synthesis from amino acids and intracellular uptake from the environment (20, 53). The intracellular levels of polyamines are tightly regulated by multiple mechanisms involving both biosynthesis and transport processes. Polyamine synthesis is regulated in part by antizyme degradation of ornithine decarboxylase, a key enzyme which decarboxylates ornithine to form putrescine (13, 41) as well as feedback inhibition by the amino acid ornithine or excess levels of polyamines (14, 45). In Escherichia coli, transcription of genes encoding a polyamine transporter is inhibited by intracellular putrescine and by excess cellular concentrations of gene products (1, 26).
Although polyamines have been shown to have multiple effects on protein synthesis and cell proliferation for all cell types, polyamine uptake and synthesis in pathogenic bacteria has not been well studied. In E. coli, a four-gene operon encodes an ABC transporter for putrescine and spermidine and a separate four-gene operon encodes a putrescine-specific transporter (21). E. coli also expresses a transmembrane protein which functions as a putrescine-ornithine antiporter (27). E. coli mutants unable to synthesize polyamines de novo grow at a much slower rate than wild-type cells (46), as do mutants which cannot transport exogenous polyamines from the immediate environment (25). Together, these findings suggested that E. coli depends upon both transport and synthesis processes to maintain optimal intracellular polyamine levels.
Most of the known details about the physiology of polyamines in prokaryotes have been derived from E. coli studies, and knowledge of polyamine metabolism and transport in other human pathogens consists only of functional genomic analysis of the completed genome projects. Many clinically relative bacterial species contain genes associated with a superfamily of amino acid-polyamine-organocation transporters as determined on the basis of sequence analysis (22), but almost nothing is known about their function during in vitro growth or in a human host. Shigella flexneri mutants unable to synthesize modified nucleosides necessary for tRNA synthesis can utilize supplemental putrescine to restore virulence gene expression (10). Enterococcus faecalis can use agmatine as another energy source and takes up this polyamine by an agmatine-putrescine antiporter (8). Putrescine and cadaverine have been found in the peptidoglycan in Veillonella and Selenomonas spp. (24).
The human pathogen Streptococcus pneumoniae (pneumococcus) is the most common bacterial cause of pneumonia, and S. pneumoniae infection can also lead to septicemia and meningitis (30). While the polysaccharide capsule of pneumococci is considered to be one of the most important virulence factors of this organism, many surface-associated and secreted proteins are now being studied for their role in pathogenesis and protective immunity (5, 23, 42). Pneumococcal genes with homology to a polyamine transporter (Pot) operon in E. coli have been implicated in the pathogenesis of pneumococcal infection in a mouse model of septicemia and pneumonia (34, 51). The sequenced genomes of pneumococcal strains TIGR4 and R6 have been published, and both strains contain the four contiguous genes potABCD that encode a putative spermidine-putrescine transporter (17, 47). Through functional genomic analysis using the National Center for Biotechnology Information BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), the function of each pot gene product was determined. The potA gene contains consensus Walker A and Walker B sites which define an ATP-binding cassette (39); PotA is therefore predicted to be a typical ATP-binding protein. The proteins encoded by potBC contain multiple -helical hydrophobic domains, a typical feature of integral membrane proteins, and are predicted to form a polyamine-specific channel through the pneumococcal cell membrane. The potD gene encodes a surface-associated spermidine- and putrescine-binding protein that has high homology to PotD in E. coli. Pneumococcal PotD contains a typical gram-positive signal peptide but does not possess motifs characteristic of lipoproteins, sortase-processed proteins, or choline-binding proteins. Figure 1 diagrams the functional genomics of the potABCD operon within Streptococcus pneumoniae. Because of the surface location and polyamine-binding function of PotD, this protein is potentially associated with pneumococcal virulence. To date, neither polyamine transport nor biosynthesis within the pneumococcus has ever been studied. In this paper, we define the actual involvement of the potD gene in polyamine transport and verify its importance in pneumococcal pathogenesis. A potD deletion mutant was created in the encapsulated, mouse-virulent serotype 3 strain WU2. Phenotype differences associated with potD inactivation within the WU2potD mutant were investigated within in vitro and in vivo growth models. The actual involvement of the potD gene in pneumococcal polyamine transport was verified by comparing the in vitro growth kinetics of WU2potD and wild-type WU2 in the presence of polyamine biosynthesis inhibitors. The importance of potD in pneumococcal pathogenesis was demonstrated in murine models of systemic and pulmonary infection. By comparing the in vitro and in vivo phenotypes of WU2potD, the involvement of polyamine transport in pneumococcal virulence was conclusively shown. This is the first time that polyamine transport has been associated with the ability of a human pathogen to cause disease.
MATERIALS AND METHODS
Bacterial strains, DNA, and growth conditions. All bacterial strains, plasmids, and PCR primers used for this work are listed in Table 1. Streptococcus pneumoniae strain WU2 expresses a serotype 3 capsule (4) and was used in all experiments. This strain is highly virulent in a mouse model of sepsis (3). To create frozen stocks of bacteria, cells were grown to early and mid-exponential phase in the rich medium Todd-Hewitt yeast extract (THY) broth at 36°C in 5% CO2 and the cells were collected by centrifugation. The cells were then washed twice in sterile phosphate-buffered saline (PBS; pH 7.0) at 4°C and stored at –80°C in PBS with 20% glycerol for bacterial stock cultures. Bacterial stocks were quantitated by counting CFU in serial dilutions plated on THY plates containing 4% sheep erythrocytes (Colorado Serum Co., Denver, CO). Bacteria from frozen stocks were used to inoculate THY or a chemically defined medium (CDM) (JRH Bioscience, Lenexa, KS) which has been previously described (50). The CDM was supplemented with filter-sterilized choline chloride, ethanolamine, spermidine, or putrescine to concentrations listed for specific experiments. For inhibition experiments, THY or CDM containing 1 mg/ml of choline was supplemented with 100 μM DFMO (-dimethyl-fluroornitithine), an inhibitor of ornithine decarboxylase (Sigma, St. Louis, MO) and 100 μM MGBG [methylgloxal-bis (guanyl hydrazone)], an inhibitor of adenosylmethionine decarboxylase (Sigma, St. Louis, MO) (2, 50, 53). For in vivo experiments, frozen stocks of WU2 and WU2potD were used to inoculate THY and the cells were grown to the exponential phase before collection by centrifugation. All cultures were grown in disposable polystyrene tubes and incubated in 5% CO2 at 36°C. Bacterial growth in liquid media was monitored by measuring the absorbance of resuspended cells at 600 nm in 1 ml cuvettes with a spectrophotometer at various time intervals during growth. All cultures were vortexed in the presence of glass beads (100 microns) (Sigma, St. Louis, MO) for two 30-s intervals to physically separate the pneumococcal cells that were growing as long viscous threads that frequently settled to the bottom of culture tubes. Desolution of the long chains was validated by gram staining before and after vortexing in the presence of glass beads. Terminal subcultures were done after all experiments by plating bacteria from the liquid media onto blood agar plates and testing for optochin sensitivity to assure purity and identity of the cultures. Bacteria concentrations (approximately 1 x 106 to 1 x 108) were estimated by spectrophotometer (A600) based on previously performed standard curves plotting A600 values against the number of viable cells for WU2 and further confirmed by viable CFU counts on sheep blood agar plates.
All DNA templates were prepared with genomic DNA isolated by collecting the bacterial cell pellet from a culture in exponential-phase growth by centrifugation and extracting the DNA as previously described (38). Growth medium and culture methods for genetic transformation were performed as previously shown (29, 35). Antibiotic concentrations in THY or blood agar plates were 100 μg/ml erythromycin for pneumococcal transformant selection or 100 μg/ml apramycin for propagation of E. coli containing pCZA342potD.
PCR amplification. All products below 5 kb were amplified using a Taq SuperPak DNA polymerase system (Sigma, St. Louis, MO), and all products above 5kb were amplified using an AccuTaq LA DNA polymerase system (Sigma, St. Louis, MO); for all amplification processes, the cycling parameters recommended by the manufacturer were used. All primers used are listed in Table 1.
Transformation and construction of mutant strain. All plasmids, primers, and S. pneumoniae strains utilized for this work are described in Table 1. To construct potD disruption vectors, an internal portion of the potD gene was amplified by PCR with intpotD1 and intpotD2 primers and ligated into suicide vector pCZA342 (Table 1). Plasmid pCZA342 (18) encodes apramycin (for selection in E. coli) and erythromycin (for selection in S. pneumoniae) resistance markers. Correct plasmid constructs (pCZA342potD) were confirmed by restriction digestion using EcoRI (Promega, Madison, WI) and PCR amplification with primers specific for the internal region of potD (intpotD1 and intpotD2 in Table 1). A S. pneumoniae RX1potD strain containing a disrupted potD gene was constructed by insertion-duplication mutagenesis with pCZA342potD according to previously described transformation protocols utilizing competence-stimulating peptide 1 (29). The RX1potD construct was confirmed by PCR with AccuTaq LA DNA polymerase (Sigma, St. Louis, MO) by use of FLpotD1 and FLpotD2 primers (Table 1). Competent WU2 cells were then transformed with chromosomal DNA from RX1potD and competence-stimulating peptide 2 as previously performed (35). The transformants were selected on 4% sheep blood agar plates supplemented with erythromycin (100 μg/ml). Each erythromycin-resistant transformant was backcrossed three times with wild-type WU2 background as previously described (28, 36). The WU2potD construct was confirmed by PCR with AccuTaq LA DNA polymerase (Sigma, St. Louis, MO) by use of FLpotD1 and FLpotD2 primers (Table 1) and by Southern blotting. Amplification of the potC gene was used as an internal control to verify that only the potD gene was disrupted by the transformation procedure in WU2potD. All mutants were stable after three 12-hour growth cycles in THY without antibiotic selection, with 100% of the 25 colonies tested retaining erythromycin resistance.
Southern blot analysis and preparation of hybridization probes. Phenol-chloroform-extracted DNA from strains WU2 and WU2potD was digested for 6 h with XbaI (Promega, Madison, WI) at 37°C according to the manufacturer's instructions. The digested DNA was subjected to electrophoresis in a 1% (wt/vol) agarose gel in Tris-borate-EDTA buffer for 15 h at a constant 24 V. Before blotting, the gel was immersed and gently agitated at room temperature in 250 mM HCl (10 min), 1.5 M NaCl-0.5 M NaOH (30 min), and 1.5 M NaCl-0.5 M Tris-HCl (pH 7.5) (30 min), with water rinses between treatments. DNA was transferred from the agarose gel to a Hybond-N+ (Amersham, Arlington Heights, Ill.) membrane by capillary blotting and was cross-linked to the membrane by using a UV cross-linker (Stratalinker; Stratagene, La Jolla, Calif.). The membrane was prehybridized with Dig EasyHyb buffer (Roche Applied Science, Indianapolis, IN) overnight and hybridized for 12 h with a digoxigenin-labeled probe made from a PCR-amplified internal region of the potD gene amplified with intpotD1 and intpotD2 primers. The PCR-amplified internal potD probe was prepared with a Dig High Prime DNA labeling system (Roche Applied Science, Indianapolis, IN). After hybridization with the probe, the membrane was washed twice in 1x SSC (SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% (wt/vol) sodium dodecyl sulfate at room temperature with gentle agitation for approximately 5 min per wash. The membrane was then washed twice with prewarmed (55°C) 0.1x SSC-1% (wt/vol) sodium dodecyl sulfate at 55°C for 15 min with gentle agitation. Visualization of the Southern blot was performed with a digoxigenin luminescent detection system (Roche Applied Science, Indianapolis, IN) following the manufacturer's instructions. Each membrane was exposed to X-ray film in a cassette for 15 min in order to visualize the location of the hybridized probe. The molecular weights of the hybridization signals were determined by comparison with a standard molecular weight ladder and a known full-length potD PCR fragment.
In vivo mouse studies. Experiments were all performed using CBA/CaHN-Btkxid (CBA/N) mice (Jackson Laboratory, Bar Harbor, Maine) and bred within the animal care facility (Veterans Affairs Medical Center, Jackson, MS). CBA/N mice have an X-linked inability to produce normal humoral antibody responses to a group of thymus-independent antigens, including type 3 pneumococcal capsular polysaccharide, making this mouse strain more susceptible to pneumococcal infections (4). A total of 1x 104 CFU/ml of either strain WU2 or WU2potD cells in PBS were injected either intraperitoneally (i.p.) or intravenously (i.v.) in a volume of 0.1 ml into 3- to 5-month-old mice for a model of systemic infection as previously described (6). For a pulmonary model of infection, pneumococci were introduced into the lungs of 3- to 5-month-old CBA/N mice by direct intratracheal (i.t.) inoculation. The animals were anesthetized by being placed for 30 s into an air-tight bell jar that contained a sterile gauze pledget moistened in 3 ml of Isoflurane (Abbot Laboratories, Abbott Park, Illinois), which was placed in the bottom of the bell jar, beneath an elevated platform preventing direct contact between the mouse and the anesthetic. After anesthesia was established, the mice were inoculated with approximately 1 x 104 CFU of WU2 or WU2potD cells. Mice were held in vertical suspension for 5 min after inoculation to facilitate deep penetration of the inoculums. For mouse survival curves, mice infected with 104 CFU of either WU2 or WU2potD cells were observed for extended time intervals and the overall survival of each mouse was recorded.
Polyamine biosynthesis inhibition assays. Wild-type WU2 and WU2potD cells were grown in separate 10-ml cultures to mid-exponential phase (optical density at 600 nm [OD600] of 0.5 to 0.6) in THY under the previously stated conditions. The cultures were collected by centrifugation, washed three times in a glucose citrate buffer (100 mM sodium citrate [pH 5.5], 2% D-glucose) to ensure an efficient removal of any contaminating exogenous polyamines, and resuspended in sterile PBS buffer (pH 7.0) to a final concentration of 106 CFU/ml of suspension. A total of 105 CFU of washed WU2 or WU2potD cells were used to inoculate 25 ml of THY or CDM supplemented with 1 mg/ml of exogenous choline and one of the following combinations: 100 μM MGBG and DFMO; 100 μM MGBG and DFMO and 50 μg/ml putrescine; or 100 μM MGBG and DFMO and 50 μg/ml spermidine. Culture growth was monitored every 2 h for 12 h by measuring culture turbidity with a spectrophotometer at OD600.
Intracellular polyamine concentration determination. Putrescine, cadaverine, and spermidine concentrations were determined by high-performance liquid chromatography as described previously (9) with some modifications. Wild-type WU2 and WU2potD cells were grown in separate 40-ml cultures to mid-exponential phase (OD600, 0.5 to 0.6) in THY or CDM plus choline with or without 100 μM MGBG and DFMO. The cultures were collected by centrifugation and washed three times in a glucose citrate buffer (100 mM sodium citrate [pH 5.5], 2% D-glucose) and twice in sterile PBS buffer (pH 7.0). The cells were resuspended in 5% HClO4, vigorously vortexed, and allowed to chill at 4°C for 12 h. After centrifugation, the supernatant was removed to a new tube. The bacterial cell extract solution was neutralized with 8.0 M NaOH to a pH of between 3 and 4 in order to precipitate the proteins. The supernatant was removed after centrifugation at 10,000 x g for 15 min at 4°C. The precipitates were washed two times with 0.1 M HClO4, and all of the supernatants were combined into one tube. The polyamines were derivatized with fluorescein-5-isothiocyanate and quantified by the capillary electrophoresis method as described previously (9).
Statistical analysis. All growth kinetics data represent experiments performed two to three times and are presented as the mean values for all samples. Error bars represent the standard error of the mean for each corresponding data set. Survival curve data were compared using Kaplan-Meier survival curves, and all statistical significance was determined using log rank tests. The MedCalc statistical program was used for all statistical analysis.
RESULTS
Generation of a potD mutant by gene disruption insertion mutagenesis. The gene for potD was disrupted by insertion duplication mutagenesis (Fig. 2A), using pCZA342 as the suicide vector that contained a cloned internal portion of the potD gene for gene targeting and an erythromycin resistance cassette for mutant isolation. The internal portion of the potD gene was obtained using PCR amplification of a 550-bp internal fragment of the potD gene, D39 chromosomal DNA, and primers intpotD1 and intpotD2 (Table 1). Initially, the potD gene was disrupted in the unencapsulated laboratory strain RX1 (data not shown). The potD-knockout mutants were isolated after transformation based on transformant resistance to 100 μg/ml of erythromycin. The resulting mutated chromosomal DNA preparations from RX1potD were used to create a potD-knockout mutant in the highly mouse-virulent capsular serotype 3 WU2 strain. Each mutant acquired from the transformation procedure was identified by resistance to 100 μg/ml erythromycin and backcrossed three times with wild-type WU2 chromosomal DNA as described in Material and Methods. The WU2potD construct was confirmed by PCR using primers specific for the full-length potD gene (FLpotD1 and FLpotD2 in Table 1; Fig. 2B) and by Southern blotting (Fig. 2C). For PCR confirmation, potD amplified from WU2potD was expected to be 7.5 kb while potD from wild-type WU2 was expected to be 1.1 kb (Fig. 2B). For the Southern blot, the expected hybridization position was 29,563 kb for WU2potD and 23,063 kb for wild-type WU2 (Fig. 2C). Both PCR and Southern blotting confirmed that WU2potD contained a plasmid insertion mutation within potD that inactivated the gene.
In vitro growth studies of WU2potD in rich medium. Growth of strain WU2potD was compared to that of the parent WU2 strain in the undefined complete medium THY and in a completely defined medium that does not contain exogenous polyamines. Growth of WU2potD was identical to that of wild-type WU2 in both THY (Fig. 3A) and in CDM-50 μg/ml choline (Fig. 3B). As expected, the growth kinetics of WU2potD in vitro were identical to those of wild-type WU2. The published pneumococcal genomes contain various enzymes predicted to be involved with the biosynthesis of polyamines from amino acids (Table 2). Because polyamine concentrations are regulated by both synthesis and transport mechanisms, the transport-deficient WU2potD strain could still maintain adequate intracellular concentrations of polyamines due to de novo synthesis of endogenous polyamines.
Effects of polyamine synthesis inhibition on WU2potD in vitro growth. Polyamine content within cells is regulated by both polyamine transport and biosynthesis. Even with potD inactivation, WU2potD would still be able to concentrate intracellular polyamines through putative de novo synthesis pathways. In order to determine how both potD inactivation and de novo polyamine synthesis inhibition would effect the growth of WU2potD, the two polyamine synthesis inhibitors MGBG and DFMO were added to either THY or CDM containing 1 mg/ml choline. For the experiment, 105 CFU of either wild-type WU2 or WU2potD were grown in THY alone or in the presence of both MGBG and DFMO (Fig. 4A). WU2 grown in the presence or absence of the inhibitors reached exponential growth at 2 h postinoculation. WU2potD grew identically to wild-type WU2 in the absence of the inhibitors. However, WU2potD grown in THY plus inhibitors had delayed growth, reaching exponential growth at 7 h postinoculation. An initial inoculum of 105 CFU of either WU2 or WU2potD was also cultured in CDM-1 mg/ml choline with or without the addition of both MGBG and DFMO (Fig. 4B). WU2 reached exponential-phase growth at 10 h postinoculation, regardless of whether the inhibitors were present or not (Fig. 4B). WU2potD grown without the addition of the inhibitors also reached exponential growth at 10 h postinoculation. Only WU2potD growth in the presence of the inhibitors MGBG and DFMO was affected (Fig. 4). WU2potD growth in medium containing both DFMO and MGBG was greatly delayed; the cells reached exponential-phase growth 16 h postinoculation. WU2 growth was not affected by the addition of MGBG and DFMO to the culture medium. WU2potD growth, however, was delayed but not prevented by the addition of MGBG and DFMO to the culture medium, indicating that the inhibition of polyamine synthesis was most important within WU2potD during the initial growth steps.
Polyamines have been shown to be involved in various steps of cell growth. Both WU2 and WU2potD were able to grow in the presence of MGBG and DFMO, although WU2potD grew much more slowly, possessing an extended lag phase compared with WU2 results. To determine whether the final intracellular polyamine concentrations for both WU2potD and WU2 were similar, the concentration of intracellular polyamines was compared for both WU2 and WU2potD. In Table 3, the intracellular concentrations of the polyamines cadaverine, spermidine, and putrescine are compared for WU2 and WU2potD grown in THY or CDM plus 1 mg/ml choline with or without MGBG and DFMO added to the medium. The intracellular levels of the all three polyamines for WU2potD grown in both THY and CDM plus 1 mg/ml choline were similar, and these concentrations did not differ much after the addition of MGBG and DFMO (Table 3). For WU2, the intracellular levels of spermidine and putrescine did not differ much for both THY and CDM plus 1 mg/ml choline with or without MGBG and DFMO (Table 3). Interestingly, the addition of MGBG and DFMO to the both mediums did increase the intracellular concentrations of cadaverine within WU2 fivefold for cells grown in THY and threefold for cells grown in CDM plus choline (Table 3).
Alternative polyamine transport systems have been previously reported for E. coli. The unaffected growth kinetics along with the increased levels of intracellular cadaverine within WU2 in the presence of MGBG and DMFO supported the idea of the existence of alternative polyamine transport systems in the pneumococcus as well. To determine whether alternative polyamine transport systems for the polyamines putrescine and spermidine could exist in WU2potD, the mutant and WU2 were grown in CDM supplemented with 1 mg/ml of choline and 50 μg/ml exogenous putrescine or spermidine with or without 100 μM of DMFO and MGBG (Fig. ). The addition of 50 μg/ml exogenous putrescine or spermidine enabled WU2potD (Fig. 5B) to display growth kinetics identical to those of wild-type WU2 (Fig. 5A), even in the presence of both MGBG and DFMO.
The decreased proliferation rate of strain WU2potD but not strain WU2 resulting from the addition of DFMO and MGBG confirmed that PotD was involved in polyamine transport. Although WU2potD did display an extended lag phase compared with wild-type WU2 in the presence of MGBG and DFMO, both strains possessed comparable concentrations of intracellular polyamines once exponential-phase growth was reached. Also, the addition of exogenous putrescine or spermidine reinstated wild-type-like growth in WU2potD in the presence of MGBG and DFMO. These findings support the existence of potD alternative polyamine transport system(s) and possible alternative de novo biosynthesis pathways within the pneumococcus.
In vivo experiments in murine models of systemic and pulmonary infection. The potD gene was previously predicted to be associated with pneumococcal pathogenesis in mouse models of septicemia and pneumonia as determined by use of signature-tagged mutagenesis (34). To verify the involvement of potD in pathogenesis, we investigated the virulence of WU2potD within systemic and pulmonary infections. For systemic infection, mice were given i.p. (Fig. 6A) or i.v. (Fig. 6B) inoculations of 104 CFU of either wild-type WU2 or WU2potD. Five mice per bacterial strain were inoculated i.p. and 10 mice per bacterial strain were inoculated i.v. with either WU2 or WU2potD. For the pneumonia model, 10 mice were infected through an i.t. route with 104 CFU of either wild-type WU2 or WU2potD (Fig. 6C). For all infection models, a statistically significant delay in time existed before the occurrence of a terminal infection in the mice inoculated with WU2potD compared to the results seen with the mice infected with wild-type WU2. The P values for all infection models were as follows: for i.p. infection, 0.0116; for i.v., 0.0122; and for i.t., 0.0052. All murine infection models indicated that WU2potD had attenuated virulence regardless of the infection route or model tested.
DISCUSSION
Previously, a possible association of the putative polyamine transport gene potD with pneumococcal pathogenesis was suggested (34). In this study, we determined the in vitro and in vivo effects associated with the creation of the potD-deficient mutant WU2potD. Because WU2potD does not produce functional PotD, the mutant strain was unable to uptake polyamines through the activities of the transport system encoded by potABCD. Therefore, WU2potD offered the opportunity to determine whether potD was involved in polyamine transport and whether it is important in pneumococcal virulence. WU2potD displayed growth kinetics identical to those of wild-type WU2 in various rich mediums, indicating that the pot operon is not necessary for in vitro growth of the pneumococcus within such environments.
Although WU2potD had in vitro growth kinetics identical to those of wild-type WU2, the in vivo growth and associated virulence of the mutant strain were vastly affected by potD inactivation. WU2potD displayed significant virulence attenuation in murine models of both septicemia and pneumonia, and polyamine transport has been well established as an integral part of cell growth and proliferation. When a pathogen invades a potential host, the pathogen must adapt quickly to a new environment to multiply and evade the host immune system. Consequently, the pathogen would have to simulate the cellular processes of transcription and translation, both of which are processes in which polyamines would be actively involved. Therefore, polyamine transport would be important for the initial steps in pneumococcal infection. Because PotD is the polyamine-binding protein of the PotABCD system, inactivation of the associated potD gene would prevent polyamine uptake via the PotABCD system and decrease the amount of available polyamines within the bacterial cell as a whole. Although most cells obtain polyamines through multiple de novo synthesis pathways and transport systems, the removal of one transport element could significantly decrease the pool of available polyamines; during a time of environmental stress such as in vivo infection, this decrease of intracellular polyamines could affect the overall proliferation and virulence of the bacteria. The inactivation of the potD gene in the pneumococcus greatly decreased the pathogenesis of the bacteria within the host, regardless of the inoculation route or whether the infection was systemic or pulmonary in nature.
Because strain WU2potD displayed in vitro growth kinetics identical to those of the wild-type WU2 strain, the involvement of PotD in polyamine uptake needed to be defined. The polyamine de novo synthesis pathways of Streptococcus pneumoniae were inhibited to determine whether the inactivation of potD affected cell proliferation in a polyamine-deficient medium. The polyamine biosynthesis inhibitors DFMO and MGBG were utilized to stop putrescine production from ornithine and spermidine synthesis. WU2potD growth delay due to the addition of the polyamine biosynthesis inhibitors directly shows the involvement of the potD gene with polyamine uptake. Interestingly, WU2potD was able to grow in medium supplemented with DFMO and MGBG only (no exogenous polyamines were added), although the growth rate was much slower than the growth rate for wild-type WU2. These data suggested that WU2potD possibly possessed alternative means of polyamine obtainment. To determine whether WU2potD was able to obtain polyamines through pathways other than PotD-associated ones, the levels of intracellular polyamines for WU2 and WU2potD were compared for both strains during exponential-phase growth. Intracellular polyamine levels for both WU2 and WU2potD were similar for putrescine, spermidine, and cadaverine, as both strains entered exponential-phase growth in THY and CDM plus 1 mg/ml choline. The addition of MGBG and DFMO to THY and CDM plus 1 mg/ml choline did not alter the exponential-phase growth concentrations of putrescine, spermidine, or cadaverine in WU2potD or the concentrations of putrescine and spermidine in WU2. However, for WU2, the intracellular concentrations of cadaverine were greatly increased by the addition of MGBG and DFMO to the growth medium. These observations suggest that alternative polyamine biosynthesis and/or transport pathways may be present within the pneumococcus. Most bacterial cells have been show to possess a secondary pathway for putrescine production that is based on the conversion of the amino acid arginine into putrescine by arginine decarboxylase (ADC) (15, 31). Streptococcus pneumoniae may contain an inducible form of ADC that could facilitate pneumococci growth in the presence of DFMO and MGBG. Inducible ADC has been shown to exist in E. coli (46). Also, the increased concentrations of cadaverine within WU2 but not WU2potD after the addition of MGBG and DFMO to both THY and CDM plus choline indicates that potD could potentially be involved in cadaverine transport. Streptococcus pneumoniae may also upregulate the transport or biosynthesis of cadaverine in response to polyamine biosynthesis inhibition. Both published pneumococcal genomes contain cad, the gene that encodes lysine decarboxylase, an enzyme which converts the amino acid L-lysine into the polyamine cadaverine. In E. coli, cells in which the biosynthesis of putrescine is blocked by mutations upregulate the activity of the lysine decarboxylase and increase the production of cadaverine, a polyamine involved in E. coli protection from various environmental stresses (12, 14, 37). The complete reinstatement of wild-type growth in WU2potD by the addition of exogenous putrescine or spermidine was also surprising. The ability of WU2potD to use exogenous polyamines suggests that another polyamine transport system may exist within Streptococcus pneumoniae. Although different polyamines have been shown to share the same transport system(s), most cells have multiple polyamine uptake systems to ensure adequate intracellular concentration of polyamines (21). Although the published pneumococcal genomes do not seem to contain polyamine-specific transport genes other than potABCD, transport systems with multiple substrate specificities have been found in other cells. The uptake of putrescine and spermidine by Saccharomyces cerevisiae occurs via the activities of Gap1p, a protein previously believed to be mainly involved in the uptake of various amino acids (48). E. coli also encodes a putrescine-ornithine antiporter which has been shown not only to export putrescine during times of excess polyamines or acidic environmental pH but also to catalyze putrescine uptake (27). In both published pneumococcal genomes, YfnA shows 81.1% alignment to PotE in E. coli with a bit score of 112 out of 281, indicating potential similarities between protein functions (32). This gene could possibly be involved in polyamine antiporter activities within pneumococci.
Streptococcus pneumoniae is known to possess a multitude of virulence factors, from its capsule to various individual proteins that are necessary for an effective infection. The expression of virulence-associated genes has been shown to be unregulated upon in vivo infection (34). This upregulation of virulence gene expression results from the summation of different environmental influences and involves both the transcription of mRNA and the translation of various proteins. The concentrations of intracellular polyamines have been shown to influence the efficiency and fidelity of translation and to stabilize DNA, RNA, and various enzymes involved in both transcription and translation as well as to modulate the synthesis of various proteins at the translational level (31). The polyamine putrescine has been shown to restore virulence gene expression in the pathogen Shigella flexneri (10), to influence the formation of filaments in Candida albicans that are necessary for virulence in mouse models (16), and to be involved in host cell adherence by Trichomonas vaginalis (11). The data presented here show that the polyamine transport system encoded by potABCD influences the overall pathogenesis of Streptococcus pneumoniae within the murine host. The virulence attenuation occurs within multiple infection models and inoculation routes, supporting the importance of the potD gene and polyamine transport during the initial stages of infection. Because polyamines appear to have multiple effects within the pneumococcus, the study of polyamine function and uptake within this human pathogen is important and warrants further study. Also, the existence of many polyamine synthesis and transport inhibitors offers endless possibilities for new approaches to preventing human infection with Streptococcus pneumoniae.
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