PhoPR Contributes to Staphylococcus aureus Growth during Phosphate Starvation and Pathogenesis in an Environment-Specific Manner

Microbial pathogens must obtain all essential nutrients, including phosphate, from the host. To optimize phosphate acquisition in diverse and dynamic environments, such as mammalian tissues, many bacteria use the PhoPR two-component system.

ABSTRACT

Microbial pathogens must obtain all essential nutrients, including phosphate, from the host. To optimize phosphate acquisition in diverse and dynamic environments, such as mammalian tissues, many bacteria use the PhoPR two-component system. Despite the necessity of this system for virulence in several species, PhoPR has not been studied in the major human pathogen Staphylococcus aureus. To illuminate its role in staphylococcal physiology, we initially assessed whether PhoPR controls the expression of the three inorganic phosphate (P_i) importers (PstSCAB, NptA, and PitA) in S. aureus. This analysis revealed that PhoPR is required for the expression of pstSCAB and nptA and can modulate pitA expression. Consistent with a role in phosphate homeostasis, PhoPR-mediated regulation of the transporters is influenced by phosphate availability. Further investigations revealed that PhoPR is necessary for growth under P_i-limiting conditions, and in some environments, its primary role is to induce the expression of pstSCAB or nptA. Interestingly, in other environments, PhoPR is necessary for growth independent of P_i transporter expression, indicating that additional PhoPR-regulated factors promote S. aureus adaptation to low-P_i conditions. Together, these data suggest that PhoPR differentially contributes to growth in an environment-specific manner. In a systemic infection model, a mutant of S. aureus lacking PhoPR is highly attenuated. Further investigation revealed that PhoPR-regulated factors, in addition to P_i transporters, are critical for staphylococcal pathogenesis. Cumulatively, these findings point to an important role for PhoPR in orchestrating P_i acquisition as well as transporter-independent mechanisms that contribute to S. aureus virulence.

INTRODUCTION

Staphylococcus aureus is a ubiquitous nosocomial organism with a diverse arsenal of virulence factors enabling it to establish infection in any mammalian tissue upon invasion (1–4). Consequently, S. aureus is a leading cause of infective endocarditis, skin and soft tissue infections, and medical device-associated infections, among other diseases (5, 6). Widespread antibiotic resistance among health care- and community-associated isolates complicates the treatment of staphylococcal disease (7, 8), prompting the Centers for Disease Control and Prevention (CDC) to name S. aureus a serious threat to human health (9). In light of the concurrent decline in the development of new antimicrobial therapies, agencies like the CDC and the World Health Organization have called for novel strategies for treating S. aureus infections (9, 10). Increasing our understanding of the aspects of microbial physiology that impact virulence will enhance our ability to identify new opportunities to treat bacterial infections.

Inorganic phosphate (P_i) acquisition is critical for microbial pathogenesis. Mutation of P_i acquisition systems decreases the virulence of S. aureus and a variety of organisms, including Escherichia coli and other Enterobacteriaceae, Vibrio cholerae, Mycobacterium tuberculosis, and Streptococcus pneumoniae (11–18). S. aureus encodes three P_i transporters that are important for growth in distinct environments and differentially contribute to staphylococcal pathogenesis (Fig. 1) (18). Two of these P_i transporters, PstSCAB and PitA, are similar to those found in E. coli, the primary model for the study of bacterial P_i acquisition. In S. aureus, the low-affinity, proton motive force-driven PitA transporter is expressed in P_i-replete media and is further induced during P_i starvation. PstSCAB is an ABC family importer expressed during P_i limitation and optimally contributes to uptake when this nutrient is scarce (18). Differing from E. coli, S. aureus possesses a third P_i transporter, NptA, that is expressed in excess P_i and is significantly induced when the amount of P_i is limiting (18). NptA is a member of the NaPi-2 family of sodium-phosphate transporters, which have been sparsely characterized in bacteria (19, 20). Interestingly, environmental factors other than P_i availability drive the efficacy of the transporters, with PitA being optimal for P_i acquisition in acidic environments and NptA being optimal in alkaline ones (18). Unlike observations in E. coli and other Enterobacteriaceae, the loss of PstSCAB does not attenuate staphylococcal virulence (18). Those studies revealed that NptA has an important role in the pathogenesis of S. aureus (18).

FIG 1.

Model of phosphate uptake and homeostasis in S. aureus. The staphylococcal two-component system PhoPR is required for the expression of the P_i transporters PstSCAB and NptA and can modulate the expression of PitA in response to low P_i concentrations. Each of these transporters is associated with a PhoU protein/domain, which has been associated with controlling the activity of PhoPR homologs in other bacteria. The PhoU homolog associated with the pitA locus is pitR. (A) In some environments, such as in alkaline, phosphate-limited growth medium or the liver, PhoPR is required to induce the expression of P_i transporters, PstSCAB and NptA. (B) In other environments, such as in neutral or acidic phosphate-limited growth medium and the heart, PhoPR is necessary to induce the expression of genes other than P_i transporters. Boxes indicate loci necessary for resisting P_i starvation under the given conditions.

The observation that the three S. aureus P_i transporters are differentially expressed as a function of P_i availability means that their expression must somehow be regulated. To regulate P_i acquisition and homeostasis, many bacteria contain a P_i-responsive two-component system (TCS) called PhoPR (PhoBR in Escherichia coli and other Gram-negative organisms, PnpRS in Streptococcus pneumoniae, and SenX3-RegX3 in M. tuberculosis) (21). The importance of maintaining proper P_i homeostasis is highlighted by the observation that deletion or disruption of PhoPR decreases the virulence of E. coli, V. cholerae, M. tuberculosis, and others (12, 22–25). In these organisms, PhoPR is activated upon P_i starvation and induces the transcription of genes involved in high-affinity phosphate assimilation, collectively called the Pho regulon. The Pho regulon of a variety of organisms (Caulobacter crescentus, Bacillus subtilis, and Streptomyces coelicolor), including human pathogens (pathogenic E. coli, Salmonella enterica serovar Typhimurium, S. pneumoniae, and Bacteroides fragilis), has been elucidated (20, 23, 26–30). Conserved, direct targets of PhoPR frequently include PstSCAB, an alkaline phosphatase, and transporters for organic sources of phosphate, such as glycerol-3-phosphate (21, 31). In addition to governing P_i acquisition, the loss of PhoPR disrupts a variety of cellular processes that critically contribute to the outcome of infection, including biofilm formation, quorum sensing, capsule production, and the response to environmental stressors such as acidic or alkaline pH (11, 12, 20, 32–35). Virulence factors, including macromolecular secretion systems, are also frequently among the genes whose expression is directly or indirectly controlled by PhoPR (29, 36–38). S. aureus encodes a homolog of the PhoPR system. Although the importance of the staphylococcal PhoPR system has not been elucidated, one study has already connected it to pathogenesis with the observation that a transposon insertion in phoP results in increased hemolysin activity (39).

PhoPR-mediated P_i sensing is best characterized in the model organisms E. coli and B. subtilis (31, 40–42). PhoR is a membrane-bound sensor histidine kinase that phosphorylates the response regulator PhoP upon P_i limitation (31). In E. coli, PhoR, PstSCAB, and PhoU, a phosphate regulatory protein encoded at the pst locus, interact at the cytoplasmic membrane to form a P_i-signaling complex (43). In E. coli and others, the PhoR/PstSCAB/PhoU complex is necessary for the repression of the TCS, and mutation of its components leads to constitutive Pho regulon expression (20, 31, 37, 44). Differing from E. coli, B. subtilis lacks a PhoU protein and does not require an intact Pst system for proper P_i signaling; rather, PhoPR senses P_i starvation through cell wall intermediates that accumulate from a blockage in the synthesis of phosphate-rich lipoteichoic acid (41, 45). Such differences illustrate that there is considerable variety in how phosphate homeostasis is sensed among bacteria.

Given the critical contribution of phosphate homeostasis to microbial physiology and recent links to antibiotic resistance (46, 47), we set out to elucidate the contribution of PhoPR to phosphate homeostasis and pathogenesis in S. aureus. Our investigations revealed that PhoPR is necessary for the expression of two S. aureus P_i transporters, PstSCAB and NptA, and influences the expression of the third, PitA. Optimal growth of S. aureus in P_i-limiting media requires PhoPR. Further investigations revealed that, depending on the environment, this requirement involves the expression of P_i transporters or P_i transport-independent mechanisms. PhoPR is necessary for wild-type levels of virulence, and PhoPR-controlled factors in addition to P_i transporters critically contribute to pathogenesis. Altogether, these findings indicate that PhoPR differentially contributes to growth in distinct environments and support an important role for PhoPR during S. aureus infection independent of P_i transporter expression.

RESULTS

PhoPR is necessary for expression of P_i transporters PstSCAB and NptA in S. aureus.

Our previous analysis of the S. aureus genome suggested that it contains a PhoPR TCS (18). To determine whether this putative PhoPR system regulates the P_i transporters of S. aureus, the expression levels of pstSCAB, nptA, and pitA were measured via transcriptional reporter fusions in a ΔphoPR mutant and compared to those of in wild type. Bacteria were grown in the phosphate-deplete defined medium PFM9 (phosphate-free M9-based medium) (18) at neutral pH (pH 7.4), supplemented with excess (5 mM) or limiting (50 μM) amounts of P_i. Consistent with our previous finding, the pst system is not expressed when there is excess P_i (Fig. 2A) (18). The expression of pstS was also not detected under these conditions in the ΔphoPR mutant (Fig. 2A). In P_i-limiting medium, while pstS was highly induced in the wild type, there was no induction in the ΔphoPR mutant, indicating that pst expression is dependent on PhoPR (Fig. 2A). In the wild type, nptA was expressed in excess P_i, and its expression was increased ∼4-fold upon P_i starvation; however, all nptA expression was abrogated in the ΔphoPR mutant, indicating that nptA expression is also dependent on PhoPR (Fig. 2A). The expression level of pitA was unchanged in the ΔphoPR mutant compared to the wild type in P_i-replete or -deplete medium, suggesting that PhoPR does not affect its expression under these conditions (Fig. 2A). Cumulatively, these data indicate that the expression of pstSCAB and nptA, but not that of pitA, is PhoPR dependent.

FIG 2.

PhoPR is necessary for expression of P_i transporters PstSCAB and NptA in S. aureus. Shown is P_i transporter expression in S. aureus wild-type (WT) and ΔphoPR strains after 9 h of growth in PFM9 buffered to pH 7.4 (A), pH 6.4 (B), or pH 8.4 (C) and supplemented with excess P_i (5 mM P_i) or a limiting amount of P_i (50 μM P_i [A and B] or 158 μM P_i [C]) and chloramphenicol for plasmid maintenance. Expression was assessed by measuring fluorescence using the P_pstS-yfp, P_nptA-yfp, and P_pitA-yfp reporter plasmids. *, P < 0.05 compared to the value for the wild type via two-way analysis of variance (ANOVA) with Sidak's multiple-comparison test (n = 3). Error bars indicate standard errors of the means (SEM).

Previously, we found that pH is an environmental factor that impacts the expression of the three S. aureus P_i transporters (18). In addition to the acidity that pathogens canonically face in the phagolysosome (48–50), some tissues, such as the blood and the skin, become alkaline upon bacterial infection, which may increase the likelihood of staphylococcal infection (51, 52). These findings led us to interrogate whether pH influences the PhoPR-mediated regulation of P_i transporter expression. Expression levels of pstS, nptA, and pitA were measured in the wild-type and ΔphoPR strains grown in acidic (pH 6.4) and alkaline (pH 8.4) PFM9. Given the observation that the expression of pst and nptA is PhoPR dependent (Fig. 2A), combined with our previous finding that a ΔpstSCAB ΔnptA strain is unable to grow in alkaline, P_i-limiting medium (18), we measured the expression levels in the strains at alkaline pH with a higher but still limiting P_i concentration (158 μM P_i) to allow for some growth of a strain lacking both pstSCAB and nptA. In both acidic and alkaline media, regardless of the P_i concentration, the expression of pstS and nptA was absent in the ΔphoPR mutant, consistent with the idea that their expression is entirely dependent on PhoPR (Fig. 2B and C). Under P_i-sufficient conditions, the expression of pitA was unchanged in the ΔphoPR mutant at either acidic or alkaline pH (Fig. 2B and C). However, in acidic, P_i-deplete medium, pitA expression decreased slightly (∼30%) but statistically significantly in the ΔphoPR mutant (Fig. 2B). Additionally, pitA expression decreased by half in the ΔphoPR strain in P_i-limiting, alkaline medium (Fig. 2C). The expression of pitA was also decreased in the ΔphoPR mutant at other limiting P_i concentrations in acidic and basic media (data not shown). These observations suggest that PhoPR is necessary for proper pitA expression during P_i starvation under acidic or alkaline conditions. Altogether, these data bolster the conclusion that PstSCAB and NptA depend on PhoPR for expression and suggest that this TCS influences the expression of PitA, depending on the environment.

PhoU is not a dominant negative regulator of PhoPR activity in S. aureus.

In E. coli and several other species, the PhoU protein has been described as a negative regulator of PhoPR (PhoBR), as disruption of phoU results in PhoPR activation and the expression of the Pho regulon (20, 31, 53). Aberrant PhoPR activation in phoU mutants of E. coli and these other species is associated with severe growth defects under P_i-replete conditions (20, 54). We previously determined that, differing from E. coli, S. aureus harbors multiple phoU homologs (Fig. 1) (18). To determine if PhoU encoded at the pst locus dominantly controls the activity of PhoPR in S. aureus, we assessed the expression of the three staphylococcal P_i transporters in a ΔphoU mutant in neutral P_i-deplete and -replete media. In P_i-deplete medium, the magnitude of transporter expression in the ΔphoU mutant was similar to that in the wild type (Fig. 3A). Remarkably, the expression of the transporters was unchanged compared to the wild type in P_i-replete medium (Fig. 3A). Consistent with this observation, the ΔphoU mutant did not have a growth defect in P_i-replete medium (Fig. 3B). Cumulatively, these findings suggest that PhoU is not a dominant regulator of the Pho regulon in S. aureus.

FIG 3.

PhoU is not a dominant regulator of PhoPR activity in S. aureus. (A) P_i transporter expression in S. aureus wild-type and ΔphoU strains after 9 h of growth in PFM9 buffered to pH 7.4 and supplemented with excess (5 mM) or limiting (158 μM) amounts of P_i and chloramphenicol for plasmid maintenance. Expression was assessed by measuring fluorescence using the P_pstS-yfp, P_nptA-yfp, and P_pitA-yfp reporter plasmids. Statistical analysis via two-way ANOVA revealed no significant differences in expression between the strains (n = 3). Error bars indicate SEM. (B) Growth of S. aureus wild-type and ΔphoU strains in PFM9 buffered to pH 7.4 and supplemented with excess (5 mM) or limiting (50 μM) amounts of P_i was assessed by measuring OD₆₀₀ (n = 3). Error bars indicate SEM and are frequently smaller than the symbols.

PhoPR is important for growth under P_i-limiting conditions independent of P_i transporter regulation.

To assess whether the PhoPR system in S. aureus is important for growth during phosphate limitation, growth of the wild type was compared to that of the ΔphoPR mutant under P_i-replete (5 mM) and P_i-limiting (50 μM) conditions at neutral pH. The ΔphoPR mutant grew similarly to the wild type when there was excess P_i (5 mM P_i) (Fig. 4A). However, under P_i-limiting (50 μM P_i) conditions, the mutant grew to a significantly lower terminal optical density than the wild type (Fig. 4B). This growth defect could be complemented by the ectopic expression of phoPR (Fig. 4C). These data support a role for PhoPR of S. aureus in resisting phosphate starvation.

FIG 4.

PhoPR is important for resisting P_i starvation independent of regulating P_i acquisition. (A and B) Growth of S. aureus wild-type, ΔphoPR, ΔpstSCAB ΔnptA, and ΔpstSCAB ΔnptA ΔphoPR strains in PFM9 buffered to pH 7.4 and supplemented with 5 mM P_i (A) or 50 μM P_i (B) was assessed by measuring OD₆₀₀. (C) Growth yield after 12 h measured by the OD₆₀₀ of S. aureus wild-type and ΔphoPR strains carrying an empty vector (pP_lgt) or phoPR (pP_lgt-phoPR) in PFM9 buffered to pH 7.4 and supplemented with 5 mM or 50 μM P_i (n = 3). Error bars indicate SEM and are frequently smaller than the symbols. (D) Expression of pitA was assessed in wild-type, ΔpstSCAB ΔnptA, and ΔpstSCAB ΔnptA ΔphoPR strains using P_pitA-yfp following growth in PFM9 containing 5 mM or 50 μM P_i buffered to pH 7.4 (n = 3). Error bars indicate SEM. *, P < 0.05 relative to the wild type via two-way ANOVA with a Tukey multiple-comparison test. (E) Growth after 12 h of the indicated strains in PFM9 supplemented with 50 μM P_i buffered to pH 7.4 (n = 3). Error bars indicate SEM. *, P < 0.05 via an unpaired t test. (F) Intracellular P_i levels in wild-type and ΔphoPR strains grown in PFM9 adjusted to pH 7.4 and supplemented with 5 mM or 50 μM P_i (n = 3). Error bars indicate SEM. Statistical analysis via two-way ANOVA revealed no significant differences in P_i accumulation between the strains. Chloramphenicol was added for plasmid maintenance, and 200 ng/ml anhydrotetracycline was added for gene induction as necessary.

To determine whether the growth defect of the ΔphoPR mutant in P_i-limiting medium was due to a loss of expression of pstSCAB and nptA, we compared the growth of the ΔphoPR mutant to that of the ΔpstSCAB ΔnptA and ΔpstSCAB ΔnptA ΔphoPR mutants in high (5 mM) and low (50 μM) P_i concentrations. When there was excess P_i, all of the mutants grew as well as the wild type (Fig. 4A). Under the P_i-limiting condition, in contrast to the growth defect of the ΔphoPR strain, the ΔpstSCAB ΔnptA strain grew similarly to the wild type (Fig. 4B). The ΔpstSCAB ΔnptA ΔphoPR mutant had a growth defect similar to that of the ΔphoPR strain in P_i-limiting medium (Fig. 4B). These findings indicate that PstSCAB and NptA are dispensable for resisting P_i starvation under these conditions. Given our observation that PhoPR can modulate pitA expression in certain environments, it is possible that the growth defect of the ΔphoPR strain may be due to decreased expression of PitA in the absence of PhoPR. To evaluate this possibility, the expression of pitA was assessed in the ΔpstSCAB ΔnptA and ΔpstSCAB ΔnptA ΔphoPR mutants. Relative to the wild type, in the ΔpstSCAB ΔnptA mutant, there was a modest but statistically significant increase in pitA expression that was ablated in the triple mutant (Fig. 4D). As an initial step to determine if this reduced expression could explain the reduced growth of a strain lacking PhoPR, pitA was ectopically expressed from an inducible plasmid in the ΔphoPR mutant. For these experiments, a concentration of the inducer that enables this construct to rescue the growth of a ΔpitA ΔpstSCAB mutant in acidic, P_i-limited medium was used (18). This overexpression did not rescue the growth of the ΔphoPR mutant (Fig. 4E). Additionally, we measured intracellular P_i levels in the wild type and the ΔphoPR mutant in neutral medium supplemented with 5 mM P_i (excess P_i) and 158 μM P_i, a concentration that is high enough to allow sufficient biomass to be harvested for the assay but at which the ΔphoPR mutant still has a growth defect compared to the wild type. Under both P_i-replete and -limiting conditions, the ΔphoPR mutant had levels of P_i comparable to those of the wild type (Fig. 4F). The latter observations suggest that despite the modest change in pitA expression, the ΔphoPR mutant does not have a defect in P_i acquisition compared to the wild type under these conditions. Altogether, these data indicate that targets of PhoPR other than P_i transporters play a critical role in growth during phosphate starvation.

PhoPR differentially contributes to growth in distinct environments.

Given our observation that pH impacts PhoPR-mediated regulation of P_i transporter expression (Fig. 2), we sought to determine whether PhoPR is important for the growth of S. aureus under acidic or alkaline conditions. The growth of the ΔphoPR mutant was compared to that of the wild type in acidic (pH 6.4) or alkaline (pH 8.4) medium supplemented with excess or limiting amounts of P_i. When grown with excess P_i (5 mM), the ΔphoPR mutant grew similarly to the wild type in acidic medium and had a mild growth defect in alkaline medium (Fig. 5A), indicating that PhoPR is not necessary for growth in acidic or basic medium in the absence of P_i limitation. When grown in P_i-limiting (50 μM P_i) acidic medium, the ΔphoPR mutant failed to reach the same terminal optical density as the wild type, suggesting that PhoPR is important for growth during P_i starvation under acidic conditions (Fig. 5B). Strikingly, in P_i-deplete alkaline medium, growth of the ΔphoPR mutant was effectively abolished, indicating that PhoPR is essential for growth under these conditions (Fig. 5B). Growth of the ΔphoPR strain could be complemented by the ectopic expression of phoPR in both acidic and basic media (Fig. 5A and B). Together, these findings indicate that PhoPR contributes to resisting P_i starvation regardless of the pH and is indeed required for growth under P_i-limiting, alkaline conditions.

FIG 5.

PhoPR contributes to growth in both acidic and alkaline P_i-limited environments. Growth after 12 h of S. aureus wild-type and ΔphoPR strains carrying an empty vector (pP_lgt) or phoPR (pP_lgt-phoPR) in PFM9 buffered to pH 6.4 or pH 8.4 and supplemented with 5 mM P_i (A) or 50 μM P_i (B) was assessed by measuring OD₆₀₀ (n = 3). *, P < 0.05 compared to wild-type pP_lgt via two-way ANOVA with Tukey's multiple-comparison test. Error bars indicate SEM. Chloramphenicol was added for plasmid maintenance.

Given that transporter-independent factors were important for the growth of the ΔphoPR mutant under neutral P_i-limiting conditions, we next assessed their importance in acidic and alkaline media by comparing the growth of the ΔphoPR mutant to those of the ΔpstSCAB ΔnptA and ΔpstSCAB ΔnptA ΔphoPR mutants under these conditions. In P_i-replete medium, the mutants grew similarly to the wild type regardless of pH (Fig. 6A). In P_i-limiting, acidic medium, while the ΔphoPR mutant had a growth defect, the ΔpstSCAB ΔnptA strain grew similarly to the wild type (Fig. 6B). The ΔpstSCAB ΔnptA ΔphoPR mutant grew similarly to the ΔphoPR mutant (Fig. 6B). Together, these data indicate that in acidic medium, as in neutral medium, there are PhoPR-controlled factors aside from the three identified staphylococcal P_i transporters that are important for growth when P_i availability is limited. In P_i-limiting medium at alkaline pH, growth of the ΔpstSCAB ΔnptA mutant, like that of the ΔphoPR mutant, was entirely abolished (Fig. 6C). Because the ΔpstSCAB ΔnptA mutant grew similarly to the ΔphoPR mutant under these growth conditions, we hypothesized that in this environment, the strain lacking PhoPR is unable to grow because pstSCAB and nptA expression cannot be induced. To test this, pstSCAB and nptA were individually expressed from an inducible promoter in the ΔphoPR strain. The ectopic expression of pstSCAB or nptA restored the growth of the ΔphoPR mutant, suggesting that the growth defect of the ΔphoPR mutant under P_i-limiting, alkaline conditions is due to the simultaneous loss of PstSCAB and NptA expression (Fig. 6D). Since we previously observed that pitA expression is significantly decreased in a ΔphoPR background under these conditions (Fig. 2C), we also ectopically expressed pitA in the ΔphoPR mutant. Consistent with our previous finding that PitA is unable to support the growth of S. aureus in P_i-limiting, basic medium (18) due to the inability of the Pit system to import P_i under such conditions (55), the induction of pitA failed to restore the growth of the ΔphoPR mutant (Fig. 6D). This observation indicates that during phosphate starvation under alkaline conditions, PhoPR is critical for the expression of P_i transporters that can function at basic pH. Cumulatively, these data suggest that for P_i-limited growth, distinct subsets of PhoPR-regulated genes are important in different environments.

FIG 6.

PhoPR differentially contributes to P_i-limited growth in distinct environments. (A) Growth after 12 h of S. aureus wild-type, ΔpstSCAB ΔnptA, ΔphoPR, and ΔpstSCAB ΔnptA ΔphoPR strains in PFM9 buffered to pH 6.4 or 8.4 and supplemented with 5 mM P_i was assessed by measuring OD₆₀₀ (n = 3). Error bars indicate SEM. (B and C) Growth of the indicated strains in PFM9 adjusted to pH 6.4 (B) or pH 8.4 (C) and supplemented with 50 μM P_i was assessed by measuring OD₆₀₀ (n = 3). Error bars indicate SEM and are frequently smaller than the symbols. (D) Growth of the indicated strains in PFM9 buffered to pH 8.4 with 50 μM supplemental P_i, chloramphenicol for plasmid maintenance, and 200 ng/ml anhydrotetracycline for gene induction. Growth was measured by assessing the OD₆₀₀ and normalized to the value for the wild type at 12 h. *, P < 0.05 compared to wild-type pP_Tet-empty via one-way ANOVA with Dunnett's multiple-comparison test (n = 3). Error bars indicate SEM.

PhoPR is important for virulence of S. aureus independent of P_i transporter regulation.

In several bacterial pathogens, PhoPR has a critical role in virulence (12, 22–25). Additionally, phosphate metabolism has been linked to antibiotic resistance (46); specifically, a point mutation in pitA increases stationary-phase tolerance of S. aureus to the clinically important antibiotic daptomycin (DAP) (47). To determine if disruption of PhoPR has a similar effect, we compared the sensitivities of the wild type and the ΔphoPR mutant to DAP. Deletion of phoPR did not change the MIC of DAP for S. aureus (data not shown). Importantly, the ΔphoPR mutant did not display increased stationary-phase tolerance to DAP compared to the wild type (Fig. 7), suggesting that a general disruption in phosphate homeostasis does not increase tolerance to antibiotic treatment.

FIG 7.

Loss of PhoPR does not increase tolerance to daptomycin. Stationary-phase cultures of S. aureus wild-type and ΔphoPR strains were incubated with or without 25× the MIC of daptomycin (which was the same concentration for both strains). Survival was assessed by measuring CFU per milliliter at the indicated time points (n = 3). Error bars indicate SEM. The limit of detection was 10³ CFU/ml. Statistical analysis via two-way ANOVA revealed no significant differences in daptomycin tolerance between the strains.

To assess the importance of PhoPR to S. aureus pathogenesis, a systemic staphylococcal abscess model of infection was used. For these assays, C57BL/6 mice were infected with S. aureus wild-type and ΔphoPR strains. Infection with the ΔphoPR mutant resulted in 3 to 4 logs fewer CFU recovered from the heart and liver than infection with the wild type, illuminating a decisive role for PhoPR in staphylococcal pathogenesis (Fig. 8A and B). Our observation that PhoPR regulates transporter-independent factors important for P_i-limited growth in vitro led us to question whether PhoPR controls transporter-independent factors that contribute to staphylococcal pathogenesis. We reasoned that if there are PhoPR-regulated factors aside from PstSCAB and NptA that are critical for infection, a triple mutant lacking phoPR, pstSCAB, and nptA would behave similarly to the ΔphoPR mutant and be less virulent than a ΔpstSCAB ΔnptA double mutant. Differing from the ΔphoPR mutant, in the heart, the ΔpstSCAB ΔnptA mutant did not have a virulence defect compared to the wild type (Fig. 8A). However, similar to the ΔphoPR mutant, the ΔpstSCAB ΔnptA ΔphoPR mutant was highly attenuated (Fig. 8A). This result suggests that PhoPR-controlled factors aside from P_i transporters predominantly contribute to virulence in this tissue. Conversely, in the liver, infection with any of the mutants resulted in decreased bacterial burdens compared to those infected with the wild type (Fig. 8B), indicating that in this organ, the induction of P_i transporters by PhoPR contributes to infection. Surprisingly, for reasons that remain unclear, the ΔpstSCAB ΔnptA ΔphoPR mutant was less attenuated than the ΔpstSCAB ΔnptA mutant. Altogether, these data indicate that PhoPR-mediated regulation of P_i transporters and transporter-independent factors contribute to the pathogenesis of S. aureus and that the contribution of PhoPR to virulence is contingent upon the specific environment.

FIG 8.

PhoPR-regulated factors, in addition to P_i transporters, are necessary for S. aureus pathogenesis. Wild-type C57BL/6J mice were infected with S. aureus wild-type, ΔphoPR, ΔpstSCAB ΔnptA, and ΔpstSCAB ΔnptA ΔphoPR strains (Δpst indicates ΔpstSCAB). Bacterial burdens in the heart (A) and liver (B) were enumerated at 4 days postinfection. P values were determined by a Mann-Whitney test; only P values of ≤0.05 are indicated. The lines indicate medians. The data are results from two independent infections (n ≥ 19 for each group).

DISCUSSION

PhoPR, the TCS that regulates bacterial phosphate homeostasis, contributes to pathogenesis in a variety of organisms; however, the importance of PhoPR in S. aureus has not been studied. In this work, we observe that PhoPR is important for growth during P_i starvation and for staphylococcal virulence. Interestingly, we found that the contribution of PhoPR to growth and virulence is dependent on the environment (Fig. 1). PhoPR is necessary for the expression of NptA, a sparsely characterized bacterial P_i transporter that is particularly important for S. aureus pathogenesis (18). PhoPR also regulates the expression of the two other staphylococcal P_i transporters as well as additional factors that promote resistance to P_i starvation. Remarkably, we also found that PhoPR-regulated factors in addition to P_i acquisition systems critically contribute to the virulence of S. aureus.

Our data indicate that PhoPR-regulated P_i acquisition and homeostasis in S. aureus differ from established models in several key ways. First, transcriptome-wide analyses of E. coli and B. subtilis indicate that their PitA homologs are not PhoPR controlled (27, 56). Indeed, decreased P_i availability is itself not sufficient to alter the expression of PitA in E. coli (57, 58). Our data indicate that in S. aureus, PhoPR influences the expression of PitA as a function of the P_i level and pH and is also necessary for the expression of NptA. The observation that nptA expression was abolished in a ΔphoPR mutant even in P_i-replete medium, wherein PhoPR would presumably be inactive, suggests that PhoPR may indirectly regulate its expression. Second, in E. coli, P_i is sensed through the PhoR/PstSCAB/PhoU complex, while in B. subtilis, it is sensed via depletion of phosphate-containing cell wall intermediates (43, 45). We cannot currently rule out the possibility that S. aureus senses levels of cell wall intermediates to measure P_i availability. However, our previous analysis indicates that the staphylococcal genome encodes a PhoU homolog at the pitA locus and a PhoU-like domain in NptA in addition to the pst-associated phoU gene (Fig. 1) (18), suggesting that S. aureus employs a PhoU-mediated P_i-sensing mechanism. Our findings indicate that unlike in E. coli and others, PhoU does not act as a dominant negative regulator of PhoPR, as disruption of phoU does not result in constitutive P_i transporter expression. Further distinguishing S. aureus phosphate regulation from that of E. coli, our previous observation that the activity of a pstS-yfp transcriptional reporter is not increased in a ΔpstSCAB mutant (18), combined with our current finding that pstS expression is PhoPR dependent, suggests that disruption of PstSCAB also does not result in constitutive PhoPR activation. Cumulatively, these findings indicate that P_i sensing in S. aureus is fundamentally different from that in well-studied models.

S. aureus is not alone in breaking the paradigm of bacterial P_i regulation established by E. coli. M. tuberculosis encodes two PstSCAB transporters and two PhoU proteins and uses these proteins in an unusual scheme to sense P_i. While only one copy of PstSCAB contributes to P_i regulation, both PhoU proteins are involved in P_i signaling through the mycobacterial PhoPR homologs (38, 44, 53, 59). S. pneumoniae also contains two pst loci, each with its own associated phoU; whereas each PhoU protein regulates the activity of its cognate Pst transporter, only PhoU2 participates in P_i signaling by interacting with the pneumococcal PhoPR homologs (20). While the multiple PhoU homologs of M. tuberculosis and S. pneumoniae are encoded by loci that also contain a PstSCAB system, the three PhoU homologs in S. aureus (18) are each located at a different P_i transporter locus (Fig. 1). This difference offers the intriguing possibility that a novel permutation of PhoPR, PhoU proteins/domains, and the three P_i transporters may contribute to P_i sensing in S. aureus.

Fundamental differences in phosphate regulation among Gram-negative, Gram-positive, and mycobacterial models could reflect unique challenges in nutrient acquisition dictated by their physiology. Additionally, they could reflect phosphate availability in the natural environments of these organisms. In the context of infection, a variety of models have found an essential role for PstSCAB in the virulence of E. coli and other members of the Enterobacteriaceae (11). Detailed analysis of the contribution of the Pst system to pathogenesis in several of these organisms suggests that constitutive activation of PhoPR resulting from mutation of pstSCAB, but not P_i transport via the Pst system per se, results in decreased virulence (11, 22, 37). This observation indicates that inappropriate Pho activation is detrimental to bacterial pathogenesis. Other studies demonstrate that disruption of PhoPR can also be detrimental to bacterial virulence (12, 22–25), including our observation that PhoPR is necessary for wild-type levels of staphylococcal pathogenesis. Notably, in this work, we observe that ΔphoPR and ΔpstSCAB ΔnptA mutants are similarly attenuated in the liver. This suggests that in some environments in the host, the PitA transporter is insufficient for P_i acquisition, and thus, PhoPR is required to induce the expression of PstSCAB or NptA. However, our observation that the ΔpstSCAB ΔnptA mutant does not have a virulence defect in the heart while the ΔphoPR mutant does indicates that in other environments, PhoPR is required during infection for the induction of transporter-independent mechanisms. Whether these additional factors relate to phosphate acquisition or include other factors necessary for virulence independent of phosphate homeostasis remains to be investigated. Notably, the current results suggest that while overaccumulation of phosphate may reduce staphylococcal sensitivity to the clinically relevant antibiotic daptomycin (47), a general disruption of phosphate homeostasis does not. In total, these observations demonstrate that S. aureus has evolved multiple mechanisms necessary for survival in the host that are dependent on PhoPR, making PhoPR an attractive target for therapeutic intervention.

During phosphate limitation, several bacteria induce a P_i-sparing response to redirect P_i usage in the cell (21, 45). Many organisms utilize polyphosphate, covalently linked orthophosphate, as a storage form of P_i that can be degraded during phosphate starvation (60). In some organisms, PhoPR transcriptionally controls the enzymes responsible for polyphosphate synthesis and degradation (23, 61). In B. subtilis, growth in low concentrations of P_i and the subsequent activation of PhoPR lead to an overhaul in the cell wall composition from phosphate-rich teichoic acid, a standard building block of the Gram-positive cell wall, to phosphate-deplete teichuronic acid as a way to redirect P_i to other critical cellular processes (45). Our observation that a ΔphoPR mutant of S. aureus has a P_i transporter-independent growth defect in P_i-deplete medium suggests that S. aureus enacts a P_i-sparing response when the amount of this nutrient is limiting. While S. aureus accumulates polyphosphate in stationary phase after growth in a P_i-rich medium (47), its propensity to accumulate polyphosphate during exponential growth and as a function of P_i availability has not been directly evaluated. However, because our assays in which the growth phenotype of the ΔphoPR mutant was observed began with cells that were starved for P_i (Fig. 4B), it seems unlikely that the wild-type strain would have accumulated sufficient polyphosphate to account for the growth differences between these two strains. Nevertheless, the possibility remains that polyphosphate metabolism in S. aureus is PhoPR regulated and important for growth during P_i starvation. S. aureus is incapable of synthesizing teichuronic acid (62) and thus cannot undergo the same change in cell wall composition as B. subtilis. Hence, there are likely as-yet-unidentified mechanisms used by S. aureus to redistribute P_i during P_i limitation.

In addition to resisting P_i starvation, PhoPR has been connected to resistance to various stressors, including nutrient starvation (21) and pH stress (11). Our analysis of the importance of PhoPR in culture indicates that in some environments, e.g., an alkaline, P_i-limiting environment, PhoPR is primarily required for induction of the Pst and NptA transporters. In others, it is essential for other unidentified factors independent of P_i transporters. Evidence in other organisms suggests that targets of PhoPR can be differentially expressed depending on the nature of the growth conditions. For example, in B. subtilis, the pst operon is specifically induced under alkaline conditions in a PhoR-dependent manner; however, other members of the Pho regulon are not expressed, and the addition of extra P_i can block pst induction (35). Those findings suggest that the high-affinity Pst system is induced due to the inability of a low-affinity transporter(s) to import P_i under alkaline conditions. This is consistent with our findings that because PitA is insufficient to support staphylococcal growth in P_i-limiting alkaline medium, either NptA or PstSCAB is required for P_i acquisition in this environment (18). Taken together with the idea that PhoPR-mediated P_i signaling can substantially differ between bacterial species, the notion that PhoPR may facilitate distinct, species-specific processes during pathogenesis provides a strong impetus for continued investigations into the role of PhoPR in staphylococcal infection.

As a postantibiotic era looms, versatile pathogens like S. aureus stand poised to reemerge as a significant cause of death. Rife with possibility for targeted disruption are regulatory networks like PhoPR that control multiple processes important for bacterial pathogenesis. The importance of investigating bacterial phosphate metabolism is further emphasized by observations that connect it to antibiotic resistance in S. aureus and other pathogens (46, 47). Further investigation into phosphate homeostasis has the potential to shed light on new ways to battle microbial invaders.

MATERIALS AND METHODS

Ethics statement.

All experiments involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois at Urbana-Champaign (IACUC license number 15059) and performed according to NIH guidelines, the Animal Welfare Act, and U.S. federal law.

Bacterial strains and cloning.

S. aureus strain Newman and its derivatives were used for all experiments. S. aureus was routinely grown in tryptic soy broth (TSB) and on tryptic soy agar (TSA) plates, while E. coli was routinely cultivated in Luria broth (LB) and on Luria agar plates. Both species were grown at 37°C; all strains were stored in media containing 30% glycerol at −80°C. As needed to maintain plasmids, 100 μg/ml ampicillin or 10 μg/ml chloramphenicol was added to the growth medium. Anhydrotetracycline was added at a concentration of 200 ng/ml for gene induction where indicated.

The S. aureus ΔphoPR mutant was generated by amplifying the 5′- and 3′-flanking regions (∼1 kb up- and downstream) of phoPR using the indicated primers (Table 1). Fragments were cloned into the pKOR1 knockout vector via site-specific recombination using the Gateway BP Clonase II enzyme mix (Thermo Fisher Scientific). The deletions were constructed via allelic exchange in Newman and the ΔpstSCAB ΔnptA mutant (18) (Table 2), as described previously (63). All mutant strains were confirmed to be hemolytic when grown on TSA blood agar plates. The S. aureus US300 (JE2) phoU::erm allele was obtained from the Nebraska Transposon Mutant Library (64) and transduced via Phi85 phage into the Newman background. For complementation studies, phoPR was cloned into the pOS1 vector (65) under the control of the S. aureus lgt promoter using the indicated primers (Table 1). pstSCAB and nptA were cloned under the control of an anhydrotetracycline-inducible promoter into the pRMC2 vector (66) (Table 3) using the indicated primers (Table 1). The pP_Tet-pitA plasmid and the pstSCAB, nptA, pitA, and empty vector reporter plasmids were generated previously (18). All PCR-generated constructs were verified by sequencing.

TABLE 1.

Primers used in this study

TABLE 2.

Staphylococcus aureus strains used in this study

Strain	Descriptiona	Reference
Newman	Wild-type S. aureus
ΔpstSCAB ΔnptA	Clean deletions of pstSCAB and nptA in Newman	18
ΔphoPR	Clean deletion of phoPR in Newman	This study
ΔpstSCAB ΔnptA ΔphoPR	Clean deletion of phoPR in the ΔpstSCAB ΔnptA strain	This study
ΔphoU	NTML phoU::erm allele phage transduced into Newman	This study

^aNTML, Nebraska Transposon Mutant Library.

TABLE 3.

Plasmids used in this study

Plasmid	Descriptiona	Reference
pKOR1	Allelic replacement vector	63
pKOR1::ΔphoPR	phoPR deletion allelic replacement vector	This study
pAH5E	YFP transcriptional reporter vector with no promoter driving yfp expression	68
pPpstS-yfp	pAH5E with pst promoter driving yfp expression	18
pPnptA-yfp	pAH5E with nptA promoter driving yfp expression	18
pPpitA-yfp	pAH5E with pit promoter driving yfp expression	18
pOS1-Plgt	Expression vector controlled by the staphylococcal lgt promoter	65
pOS1-Plgt::phoPR	phoPR in pOS1-Plgt	This study
pPTet-empty	pRMC2 (anhydrotetracycline-inducible expression vector)	66
pPTet-pstSCAB	pstSCAB in pRMC2	This study
pPTet-nptA	nptA in pRMC2	This study
pPTet-pitA	pitA in pRMC2	18

^aYFP, yellow fluorescent protein.

Growth medium, phosphate growth assays, and expression analysis.

Phosphate-free M9-based medium (PFM9) was used for growth assays and was described previously (18). Briefly, phosphate-free M9 salts were supplemented with 70 mM MOPS (morpholinepropanesulfonic acid) (for pH 6.4), HEPES (for pH 7.4), or Tris (for pH 8.4); trace amino acids; trace vitamins; 0.5% glucose; 6.2 mM β-mercaptoethanol; 2 mM MgSO₄; 1 mM CaCl₂; and 1 μM FeSO₄, ZnSO₄, and MnCl₂ to constitute PFM9 medium. P_i source stocks were made by mixing NaH₂PO₄ and Na₂HPO₄ and adjusting to the desired pH and then added to the medium to achieve the indicated P_i concentration.

For phosphate limitation growth assays, bacteria were inoculated into 5 ml TSB for 8 h and then back-diluted 1:10 into 5 ml PFM9 plus 70 mM HEPES and 158 μM P_i (pH 7.4) for 12 h. Cultures grown overnight were inoculated at a 1:100 dilution into a 96-well round-bottom plate containing 100 μl/well PFM9. Plates were incubated at 37°C with shaking at 180 rpm. Bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀). Expression (RFU [relative fluorescence units]) was determined by measuring fluorescence (excitation/emission wavelengths of 505/535 nm), normalizing the values to the OD₆₀₀, and then subtracting the RFU of empty vector controls.

Phosphate accumulation assays.

Phosphate accumulation assays were performed as described previously (18). Briefly, bacteria were grown as described above for the phosphate growth assays at the indicated pH and P_i concentration and harvested at similar optical densities (OD₆₀₀ of ∼0.2 to 0.25). Cells were washed once with and then lysed in Tris-EDTA (TE) buffer by mechanical disruption. Lysates were centrifuged to remove particulate matter and then treated with yeast exopolyphosphatase to digest any polyphosphates in the cell. The P_i concentration was measured with the Biomol green kit (Enzo Life Sciences) according to the manufacturer's instructions and normalized to the total protein concentration as measured by using the Pierce bicinchoninic acid (BCA) assay kit.

Daptomycin sensitivity assays.

Daptomycin was purchased from Acros Organics (catalog number AC461371000). The MIC of daptomycin was determined by using a standard broth microdilution assay essentially as described previously (67). Briefly, cells were grown for 16 h in TSB and then seeded at ∼5 × 10⁴ CFU/100 μl into a microtiter plate containing TSB plus 1.25 mM CaCl₂ with serial 2-fold dilutions of daptomycin. The MIC was measured in triplicate and was the same for the S. aureus wild-type and ΔphoPR strains. Daptomycin tolerance was assessed essentially as described previously (47). Briefly, cells were grown for 16 h in TSB, and 2 ml of culture was then aliquoted into 15-ml conical tubes with 1.25 mM CaCl₂ with or without daptomycin treatment at 25× the MIC. Cells were incubated at 37°C with shaking, and survival (CFU per milliliter) was measured via dilution plating at the indicated time points.

Animal infections.

Mouse infections were performed essentially as described previously, with minor modifications (18). Briefly, S. aureus wild-type, ΔphoPR, ΔpstSCAB ΔnptA, and ΔpstSCAB ΔnptA ΔphoPR strains were grown in TSB for 3 h on a roller drum; washed and resuspended in phosphate-free, carbonate-buffered saline; and diluted to a density of ∼5 × 10⁷ CFU/ml. Nine-week-old female C57BL/6J mice were injected retro-orbitally with 5 × 10⁶ CFU in 100 μl buffer. The infection was allowed to proceed for 96 h before the mice were sacrificed. Livers and hearts were removed, the organs were homogenized, and bacterial burdens were determined by plating serial dilutions.