Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus

Abstract

The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of ∼400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.

Staphylococcus aureus causes a variety of infections in humans, ranging from minor skin and wound infections to life-threatening diseases (31). The pathogenesis of staphylococcal infections is a multifactorial process that depends on the expression of different virulence factors controlled by multiple regulatory systems in conjunction with environmental and nutritional signals (46). The high degree of variability in the expression of virulence genes is modulated by a complex network regulated by factors such as the agr locus (RNAIII), SarA, and SigB (5, 9), which allows the bacterium to adapt to changing environmental conditions for survival and developing infection.

The species epithet of S. aureus reflects its characteristic surface pigmentation (aureus, meaning “golden” in Latin) (43). The yellowish-orange (golden) pigment produced by S. aureus has been linked to virulence, owing to its antioxidant property (29, 30). The golden pigmentation of S. aureus is the product of a C₃₀ triterpenoid carotenoid biosynthesis pathway, and the carotenoid pigment biosynthesis genes are organized in an operon crtOPQMN controlled by the alternative sigma factor SigB (3, 39). Since many virulence genes are coordinately regulated in S. aureus (5, 9, 31), we hypothesized that genes affecting pigmentation may also influence the production of virulence determinants and have an impact on the pathogenesis of S. aureus.

Herein, we present an analysis of S. aureus golden pigment biosynthesis and regulatory pathways at the genomic level by screening the Phoenix (ΦNΞ) library, a collection of defined transposon insertions into 1,812 open reading frames of S. aureus strain Newman (1). This study indicates an intimate link between metabolic processes and virulence gene expression. It demonstrates the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival and pathogenesis of S. aureus. Our results show that targeting purine biosynthesis is a promising strategy to develop anti-S. aureus therapies.

MATERIALS AND METHODS

Bacterial strains and chemical reagents.

S. aureus strain Newman is a human clinical isolate (13) that stably maintains an agr⁺ phenotype, i.e., quorum-controlled toxin secretion and the ability to cause animal disease (1, 37). S. aureus was propagated in tryptic soy broth ([TSB] Difco) with shaking at 250 rpm or on tryptic soy agar ([TSA] Difco). The transposon mutant collection, the Phoenix library (ΦNΞ), used in this study was described previously (1). Adenine (Acros Organics) was dissolved in 0.05 M HCl, and the same amount of HCl was used as a control. 6-Thioguanine ([6-TG] Sigma-Aldrich) and mycophenolic acid ([MPA] AK scientific, Inc.) were dissolved in dimethyl sulfoxide (DMSO) or 0.1 M NaOH, followed by neutralization with HCl. Amino acid requirements for growth were determined by growth with chemically defined medium (HHW, for Hussain, Hasting, and White) medium (18).

Phage transduction and determination of transposon insertion sites.

To determine whether unrelated mutations gave rise to the observed pigment production phenotype, all the mutant S. aureus strains used in this study were lysed with phage Φ85, and transposon insertions were transduced into wild-type strain Newman. The transduced bursa aurealis insertion sites were again mapped by PCR and DNA sequencing. All experiments were performed as described previously (1).

Measurement of carotenoid pigment and thin-layer chromatography (TLC) analysis.

Colonies of S. aureus Newman and its isogenic mutants were washed from the TSA plate and subjected to methanol extraction (36). The optical density at 465 nm (OD₄₆₅) was measured and normalized in reference to the pigment isolated from strain Newman, which was set at 100. Pigment crude extracts were separated on reversed-phase separation plates (RPSF, catalog number 52031; Analtech) with methanol-acetonitrile (1:3, vol/vol).

RNA isolation and Northern blotting.

Bacterial strains were grown in TSB or on TSA plates (a 20-μl overnight culture was plated) before being harvested for RNA extraction. The harvest and cell lysis follow the enzymatic lysis procedure described in the RNAprotect Bacteria Reagent Handbook (40) and with mechanical disruption (Fast Prep FP120 instrument; Qbiogene, Heidelberg, Germany). An RNeasy Mini Kit (Qiagen) was used for subsequent RNA purification. To isolate the RNA for microarray analysis, all S. aureus strains were grown at 37°C overnight in TSB, diluted 200-fold in 10 ml of fresh TSB in a 50-ml conical tube (BD Biosciences), and incubated at 37°C with shaking at 250 rpm for 48 h (OD₆₀₀ of 8.5 to 10). Two volumes of the RNAprotect Bacteria Reagent (Qiagen) were added to 1 volume of cell culture and treated at room temperature for 30 min. Total RNA was isolated by using an RNeasy Mini Kit (Qiagen) as described above, except that cells were lysed with 0.5 mg of lysostaphin (Sigma) per ml for 1 h at 37°C. RNA concentration and purity were determined by absorbency at 260 and 280 nm. Northern blotting was performed following previously reported procedures (6). Primers used for amplification of DNA fragments in Northern blotting are listed in Table S1 in the supplemental material. Briefly, the primers were crtM-FNF/crtM-FNR for the crtM gene, sigB-FNF/sigB-FNR for the sigB gene, sarA-FNF/sarA-FNR for the sarA gene, RNAIII-FNF/RNAIII-FNR for RNAIII, qoxB-chF/qoxB-chR for the qoxB gene, Cit-chF/Cit-chR for the citZ gene, SAV1073FNF/SAV1073FNF for the purH gene, clpXFNF/clpXFNR for the clpX gene, saeSFNF/saeSFNR for the saeS gene, lexAFNF/lexAFNR for the lexA gene, codY-FNF/codY-FNR for the codY gene, and norA_1/norA_2 for the norA gene.

Construction of plasmids for complementation of pigment mutants.

The previously constructed shuttle plasmids pYJ335 (20) and pKOR1 (2) were used in this study. To construct pYJ335::citZ, a 3,143-bp DNA fragment containing SAV1695-SAV1694 was amplified from strain Newman genomic DNA by using the primers FC-citZF and FC-citZR. To construct pYJ335::ctaA, a 2,121-bp DNA fragment containing SAV1115 was amplified by using the primers SAV1115FCR and SAV1115FCR. To construct pYJ335::SAV0672, the LysRFCF2/LysRFCR2 primer pair was used. DNA fragments were cloned into pYJ335 as described previously (8). To construct pYJ335::SAV1117 and pYJ335::purA, primer pairs SAV1117F/SAV1117R and PurAioF/PurAioR were used, respectively. In this study, the primer pairs tetRFor399/SAV1117R and tetRFor399/PurAioR were used to select the plasmid clones in which SAV1117 and purA are located downstream of the xyl-tetO promoter as described previously (7, 20). To construct pKOR1::qoxB, qoxABCD was amplified from Newman's genomic DNA by using the primers FC-qoxBF and FC-qoxBR and cloned to pKOR1 as described previously (2). The selected plasmid clones were sequenced to confirm that no additional mutations were introduced by PCRs. The correct plasmids were transformed into RN4220 and then into the mutant strain by electroporation. The pYJ335 vector or pKOR1 alone was transformed into RN4220 and then into the mutant strain and wild-type Newman as the control.

Semiquantitative plate assays for detection of hemolysins and proteases.

Briefly, hemolytic activity was analyzed on 5% sheep blood agar plates. Protease secretion was measured on 2% skim milk-TSA plates. Positive reactions, as indicated by the clearing halo surrounding the bacterial colonies, were determined 24 to 48 h following inoculation at 37°C.

Murine infection and abscess formation.

All S. aureus strains were grown at 37°C overnight in TSB, diluted 100-fold in fresh medium, and incubated at 37°C for 2.5 h until the cultures reached and OD₆₀₀ of 1.0. Bacteria were collected by centrifugation, washed, and suspended in phosphate-buffered saline (PBS) buffer. Viable staphylococci were enumerated by colony formation on TSA plates to quantify the infectious dose. A total of 100 μl of bacterial suspension (1 × 10⁷ CFU) was administered intravenously via retro-orbital injection into each of 10 6-week-old BALB/c mice. For experiments testing the efficacy of 6-TG, mice were randomized into two groups (10 mice/group) at the start of the experiment and administered 50 μl of either 8 mg/ml 6-TG (dissolved in 0.1 M NaOH, followed by neutralization with HCl) or water (supplied with the same amounts of NaOH and HCl) as a control on the day of infection by intraperitoneal injection. After 30 min of treatment, mice were challenged intraperitoneally with 100 μl of 1 × 10⁹ CFU of exponential phase S. aureus Newman per ml. For experiments testing the efficacy of adenine in reversing the attenuation of virulence of a purA mutant, infected mice (intraperitoneal injection) were repeatedly treated with subtoxic amounts (19) of adenine (total of 1 mg in 4 days with 4 injections; dissolved in 0.05 M HCl, followed by neutralization with NaOH) by intraperitoneal injection or with water (supplied with the same amounts of NaOH and HCl) as a control. Five days after injection, animals were sacrificed by CO₂ asphyxiation, and organs were removed. The organs were homogenized in 1 ml of PBS, and dilutions of the homogenates were plated on TSA. A Student's t test was performed for statistical analysis using Microsoft Excel.

Microarray hybridization and data analysis.

RNA was converted to cDNA, and microarray analysis was performed according to the manufacturer's instructions for antisense prokaryotic arrays (Affymetrix). To ensure reproducibility, three RNA samples (biological replicates) from each strain were used. Each RNA sample was independently hybridized to a GeneChip. For comparisons of each strain, GeneChip signal intensity values were normalized to the total microarray signal, averaged, and compared using GeneSpring GX, version 7.3.1, software (Agilent Technologies), as previously described (7). Genes with at least a 2.5-fold difference (P < 0.1; t test) in mRNA levels between the wild-type strain and the mutant strains were considered differentially expressed. Cluster and TreeView analysis were performed as described previously (14).

RESULTS

Screening for S. aureus pigment mutants.

The Phoenix (ΦNΞ) library (1) permits the screening of approximately two-thirds of the Newman genome. A total of 1,812 Phoenix isolates along with the isogenic wild type were grown on TSA medium at 37°C for 36 h. Variants with increased or decreased pigment production were visually identified and screened a second time in a similar fashion. Twenty-three positive candidates were grown to generate a lysate using bacteriophage Φ85 for transduction experiments. Each allele yielding altered pigment production was crossed back into strain Newman using an erythromycin resistance marker of the transposon cassette. This was performed to ensure that the phenotypes observed were not caused by any additional mutation.

Phenotypic classification of pigment mutants.

The 23 pigment mutants can be arranged in seven categories (Table 1). Ten mutants exhibited reduced pigment production while 13 displayed enhanced pigment production. Mutations in all genes in the crtOPQMN operon as well as genes in the rsbUVW-sigB system generated strains with severely decreased pigmentation (Table 1 and Fig. 1), consistent with previous reports (17, 26, 30, 39, 49). In addition, we discovered that insertions in genes ispA and nusB resulted in a dramatically decreased golden pigmentation (Table 1 and Fig. 1). To confirm these observations, we quantified the methanol-extractable carotenoid pigment from strains carrying insertions in the sigB, crtM, and ispA genes. The results confirmed that these mutant strains produce at least 3-fold lower levels of the carotenoid pigment than the wild-type Newman strain (Fig. 1B).

TABLE 1.

S. aureus mutants with reduced or enhanced pigmentation

Group, function, and strain	Pigmentationa	Gene name or description
Reduced pigmentation mutants
crtOPQMN operon
SAV2561 strain	−	crtN
SAV2562 strain	−	crtM
SAV2563 strain	−	crtQ
SAV2564 strain	−	crtP
SAV2565 strain	−	crtO
rsbUVW-sigB system
SAV2064 strain	−	sigB
SAV2066 strain	−	rsbV
SAV2067 strain	−	rsbU
Other functions
SAV1521 strain	−	ispA
SAV1524 strain	−	nusB
Enhanced pigmentation mutants
TCA cycle
SAV1695 strain	+++	citZ
SAV1851 strain	++	citG
SAV2365 strain	++	Malate:quinine oxidoreductase
Oxidative phosphorylation
SAV1115 strain	++	ctaA
SAV1060 strain	++	qoxB
Purine biosynthesis
SAV1072 strain	+	purN
SAV1073 strain	+	purH
SAV1074 strain	+	purD
SAV0017 strain	+	purA
Other functions
SAV0672 strain	+	LysR family protein
SAV1667 strain	++	hemL
SAV1108 strain	++	Hypothetical protein
SAV1117 strain	++	Hypothetical protein

^a−, reduced pigmentation observed on TSA plates compared to the wild-type Newman strain; +, enhanced pigmentation observed on TSA plates compared to the wild-type Newman strain. Additional plus signs indicate greater relative enhancement of pigmentation.

FIG. 1.

Pigmentation of representative S. aureus pigment mutants and complementation experiments. (A) Pigmentation display of S. aureus strains that were grown on a TSA plate at 37°C for 24 h. (B) Measurement of the carotenoid pigment of different S. aureus strains by methanol extraction. The relative optical density units at 465 nm were normalized to the wild-type Newman strain, which was set at 100. Results are means + standard error of the means (errors bars) of a triplicate experiment. (C) Complementation experiments of representative S. aureus mutants, as indicated. Pigmentation of S. aureus strains grown on TSA plates at 37°C for 24 h is shown. Wild-type Newman, mutant strains, and mutant strains harboring complementation plasmids (-C) are shown.

Most interesting is the finding that the 13 mutant strains not previously reported displayed enhanced pigment production (Table 1 and Fig. 1). In this case, transposon insertion occurred in several genes encoding enzymes of the tricarboxylic acid cycle (TCA) cycle (citZ, citG, and SAV2365), respiratory chain (ctaA and qoxB), and purine biosynthesis pathway (purA, purN, purH, and purD). In addition, insertions in a putative LysR family protein-encoding gene (SAV0672), an hemL gene (SAV1667), and two genes encoding two hypothetical proteins (SAV1108 and SAV1117) led to enhanced pigment production. These variants exhibited a strong yellow-orange pigmentation when plated on TSA and produced increased levels of carotenoid pigment, as quantified by methanol extraction (Fig. 1B). The carotenoid extracts derived from the wild-type Newman strain and representative S. aureus pigment mutant strains (citZ, qoxB, SAV1117, and purA strains) were further separated on RPSF plates (Analtech). As a result, all the strains exhibited similar TLC (thin-layer chromatographic) patterns (see Fig. S1 in supplemental material). Thus, it is less likely that the enhanced pigmentation is caused by a chemical modification.

To support the involvement of the TCA cycle, oxidative phosphorylation, and purine biosynthesis in pigment production, we performed complementation experiments with the citZ, ctaA, qoxB, and purA mutants. In addition, we also performed complementation experiments with SAV0672 and SAV1117 mutants. In all cases complementation of the pigment mutant with complementing plasmid restored pigment production to normal levels (Fig. 1C).

To test whether the enhanced yellow-orange pigmentation is a consequence of compromised growth, we used an approach with chemically defined medium (HHW) (18) in which a single amino acid was omitted to reveal growth-limiting amino acids. We found that the limitation of a single amino acid generally had little or no effect on the growth of the Newman strain. The main exceptions to this are proline, alanine, valine, leucine, and arginine, the limitation of which resulted in very poor growth. However, we did not observe any enhanced pigmentation when the wild-type Newman strain was grown in HHW medium with the omission of a growth-limiting amino acid.

Analysis of the expression of crtM in representative S. aureus pigment mutants.

To test whether the expression level of the carotenoid biosynthesis crtOPQMN operon was altered in some of the mutants, we performed Northern blot analysis on total RNA isolated 24 h after bacterial growth on TSA plates and examined crtM transcript levels. Seven mutants (citZ, ctaA, purH, SAV0672, sigB, ispA, and crtM strains), representing groups listed in Table 1, were analyzed. The result showed that the transcript levels of crtM correlate with changes in pigment production in four mutants (ctaA, purH, SAV0672, and sigB strains) (see Fig. S2A in the supplemental material). The crtM mutant strain exhibits the expected nonpigmented phenotype as described previously (30, 39, 49), and the truncated crtM mRNA was detected (see Fig. S2A). Mutation of ispA, a putative geranyltranstransferase gene, generated a nonpigmented mutant but showed an elevated level of crtM compared to the wild-type strain (see Fig. S2A).

Inactivation of citZ, a gene encoding the first enzyme in the citric acid cycle, resulted in increased pigment production when cells were grown on TSA plates (Fig. 1A and Table 1). However, Northern blot analysis did not reveal any significant increase in the crtM mRNA level in the citZ mutant (see Fig. S2A in the supplemental material). We further examined the crtM mRNA level in citG and SAV2365 mutant strains by Northern blot analysis. Again, there was no significant increase in the crtM mRNA level in the citG or SAV2365 mutant strain compared to that in the wild-type strain when bacteria were grown on a TSA plate, despite the increased pigment production observed in these mutants (see Fig. S2B in the supplemental material). In addition, Northern blot analysis indicates that there was no significant change in the crtM level in a qoxB mutant compared to the wild-type level when cells were grown on TSA plates (24 h), despite the increased pigment production (see Fig. S2B).

In vivo survival of pigment mutants.

Since the yellowish-orange (golden) pigment produced by S. aureus has been linked to virulence (29, 30), we next studied the in vivo survival of some pigment mutant strains by using a murine abscess model of infection (1). Interestingly, we found that the disruption of ctaA, qoxB, or purH leads to significant reduction of bacterial survival in vivo although the disruption of ctaA, qoxB, or purH gives elevated levels of pigmentation. As shown in Fig. 2, bacterial loads in the livers of mice infected with the qoxB, ctaA, or purH mutant were dramatically reduced after 5 days. Compared with the Newman strain, ctaA, qoxB, and purH mutant strains showed a >3.7-log reduction (P < 1 × 10⁻¹⁰, t test) of virulence in the livers of the infected mice (Fig. 2). The in vivo survival of the purH mutant was the most striking. This strain exhibited a 5-log reduction of bacterial loads in the liver or kidney compared to wild-type Newman. Notably, 9 of 10 animals infected with this strain had completely cleared staphylococci from the kidney after 5 days (Fig. 2). We further studied the in vivo survival of the purA mutant and found that the recovery of the purA mutant strain was reduced almost 5 logs in the kidneys of animals compared to that of wild-type Newman strain (Fig. 2C). Taken together, these results indicate that the pathogenesis of staphylococcal infections is multifactorial, given that the pigment produced by S. aureus has been linked to virulence (29, 30).

FIG. 2.

Virulence of wild-type strain Newman and ctaA, qoxB, purH, and purA mutants. The murine abscess model of infection was used to compare the virulence of strain Newman and purH, ctaA, and qoxB mutants (A and B) and of a purA mutant (C). S. aureus colonization in murine liver or kidney was measured by tissue homogenization, dilution, and colony formation on TSA plates after 5 days of infection following retro-orbital injection. Each circle indicates one mouse. Ten animals were tested for each strain. The horizontal black line represents the mean log₁₀ CFU (y axis). The statistical difference between mutant and wild-type strains was determined by a Student's two-tailed t test. The limit of detection for organ infection was 100 CFU/organ.

Characterization of pur mutants.

As described above, disrupting purine biosynthesis leads to a severe reduction of bacterial in vivo survival, and this result is consistent with the recent findings that adenosine is critical for S. aureus to escape host immune responses (47). Thus, we examined the possibility that purine biosynthesis may influence pigment production as well as the expression of other virulence determinants. purN, purH, purD, and purA mutants display increased pigmentation compared to the wild-type Newman strain; further enhanced pigmentation was observed in a purA mutant when bacteria were grown to late stationary phase (48 h) (see Fig. S2D in the supplemental material). Moreover, all four pur mutant strains exhibited decreased proteolytic activity compared to the wild type, as shown by the width of the cleared zone around colonies grown on a TSA plate containing 2% skim milk. Notably, disrupting purA resulted in a protease-defective phenotype (see Fig. S2D). These results suggest that purN, purH, and purD are functionally upstream of purA. While there was no significant change in the hemolytic activity of purN, purH, and purD mutants compared to the wild-type Newman strain, the purA mutant showed a growth defect on the TSA plate containing 5% sheep blood (see Fig. S2D).

To investigate whether disrupting purine biosynthesis affects bacterial growth in TSB medium, overnight cultures of the mutant strains were diluted in TSB medium and incubated at 37°C with shaking. The OD₆₀₀ was measured as a function of time. As shown in Fig. S3 in the supplemental material, growth of the purN, purH, or purD mutant was slightly slower than that of the wild type in log phase, and all of these mutants reached stationary phase with total growth similar to that of the wild type. In contrast, the purA mutant showed the same growth rate as the wild type in the early log phase but reached the stationary phase with a lower total growth than the wild type.

purA affects virulence gene expression.

Since purA is located downstream of the other three pur mutants (purN, purH, and purD) and exhibits the most dramatic phenotypes (see Fig. S2D in the supplemental material), we chose a purA mutant for further analysis. Complementation of the purA mutant with the pYJ335::purA plasmid fully restored proteolytic activity; the level of bacterial growth on skim milk or blood agar plates was comparable to that of the wild-type Newman strain (Fig. 3 A). Northern blot analysis revealed that the purA mutant (24 h after growth on a TSA plate) had significantly increased levels of sarA, sigB, and RNAIII mRNAs relative to the wild type (Fig. 3). The upper sarA transcript on the gel showed higher intensity than the wild type, while the lower band remained the same (Fig. 3B). The upper transcript likely originated from P3, a sigB-dependent promoter (12, 33). It is well known that sigB and sarA are negative regulators of the production of protease in S. aureus (5, 9, 25, 26). Thus, the elevated levels of sarA or sigB mRNAs may contribute to the protease-defective phenotype of the purA mutant. Although the RNAIII level is dramatically enhanced in the purA mutant, we did not observe a significantly increased production of alpha toxin in this mutant. Since PurA is an enzyme used by the bacterium to produces adenine, we examined the complementation of a purA mutant with adenine. As shown in Fig. 3C, adenine was able to complement the expression of RNAIII and sigB associated with mutation of the purA gene. To determine whether adenine could reverse the attenuation of virulence of the purA mutant (Fig. 2), infected mice were repeatedly treated with subtoxic amounts (19) of adenine (total of 1 mg in 4 days with 4 injections). Unexpectedly, the injections of adenine were not sufficient for the purA mutant to multiply in vivo (see Fig. S4 in the supplemental material).

FIG. 3.

Analysis of purA mutants. (A) Bacterial strains were inoculated onto TSA plates, TSA-2% skim milk plates, or TSA-5% sheep blood plates and incubated at 37°C for 48 h. (B) Northern blot analysis of the transcripts of sarA, sigB, and RNAIII when S. aureus strains were grown on TSA plates at 37°C for 24 h. (C) The transcripts of sigB and RNAIII were measured for the Newman and purA mutant strains grown on TSA plates (48 h) in the absence or presence of exogenous adenine (final concentration of 0.36 mM). (D) Pigmentation displays of S. aureus strains grown in TSB medium (48 h) with or without exogenous adenine, as indicated. Newman and purA mutant strains were grown on TSA-5% sheep blood plates or TSA-2% skim milk plates with or without exogenous adenine at 37°C for 48 h. Adenine concentration was set at 0.9 mM. For Northern blot analysis, 5 μg of total cellular RNA was used in each experiment, and the ethidium bromide-stained gel of the loaded RNA sample is shown below each lane.

We next evaluated the expression of selected genes after bacteria were grown to exponential (3 h), postexponential (6 h), early stationary (12 h), stationary (24 h), and late stationary (48 h) phases. Both sigB and RNAIII were included in the study, as well as crtM, a gene essential for pigment biosynthesis in S. aureus. Mutation of purA had no significant effect on the expression of crtM, sigB, or RNAIII when bacteria were grown to exponential phase (Fig. 4). However, mutation of purA led to decreased crtM expression when bacteria were grown to postexponential (6 h) or late stationary phase (48 h) but an elevated level of crtM mRNA when bacteria were grown to stationary phase (24 h). A similar expression pattern was also observed for sigB, with the exception that a decreased level of crtM transcript was observed in early-stationary-phase cells (12 h) while increased sigB expression was seen in the purA mutant compared to the wild type. Enhanced expression of RNAIII also occurred in the purA mutant compared to the wild type when bacteria were grown to stationary phase (12 h) (Fig. 4). These results show that the purA mutation affects the expression of global regulators such as sigB and RNAIII and that the effects are growth phase dependent.

FIG. 4.

Northern blot analysis of the transcripts of crtM, sigB, and RNAIII. The overnight culture was diluted 1,000-fold in fresh TSB medium. Bacteria were harvested after incubation at 37°C with shaking at 250 rpm for 3, 6, 12, 24, and 48 h and then subjected to total RNA isolation. Lanes 1, Newman/pYJ335; lanes 2, purA/pYJ335; lanes 3, purA/pYJ335::purA.

Inhibition of purine biosynthesis with small molecules.

To confirm the critical role of purine biosynthesis in regulating pigment production and virulence determinants, two known inhibitors of purine biosynthesis, 6-thioguanine (6-TG) and mycophenolic acid (MPA), were employed to test the inhibition of purine biosynthesis in S. aureus Newman. Enhanced pigmentation was observed when Newman was plated on TSA plates in the presence of 6-TG (Fig. 5A). Quantification of pigment production by extracting carotenoid products confirmed that both inhibitors afforded enhanced pigmentation in strain Newman (Fig. 5B). The sigB mutant displayed a colorless phenotype, and both 6-TG and MPA failed to enhance pigmentation of this mutant strain (data not shown), indicating that sigB is required for pigment production. Inhibition of purine biosynthesis in strain Newman also led to increased sigB, sarA, and RNAIII transcripts, as also observed with the purA mutant (Fig. 3B). While the P3 promoter of sarA is controlled by SigB, the increase in RNAIII is likely mediated in a SigB-independent manner since the same change was observed in Newman and sigB mutant strains after treatment with 6-TG or MPA (Fig. 5C). In addition, S. aureus Newman showed a 1.5-fold longer doubling time of exponential growth when TSB medium was supplied with 20 μg/ml 6-TG, and enhanced pigmentation was also observed (data not shown). Finally, we tested the effect of 6-TG on the in vivo survival of S. aureus in the murine abscess model. Mice treated with 6-TG exhibited a 1.5-log (31.6-fold) reduction (P < 0.008, t test) of bacterial loads in both liver and kidney (Fig. 5D and E).

FIG. 5.

Inhibition of the purine biosynthetic pathway in S. aureus. (A) Pigmentation displays of the Newman strain grown on TSA or TSA with 6-TG plates (TSA plates supplied with 10 μg/ml of 6-TG dissolved in DMSO) at 37°C for 48 h. TSA plates were supplied with the same amount of DMSO as a control. (B) Measurement of the carotenoid pigment by methanol extraction. Newman cells were harvested after growth at 37°C for 48 h on TSA plates, TSA-MPA (TSA plates supplied with 400 μg/ml of MPA dissolved in DMSO) plates, or TSA plates plus 6-TG. TSA plates were supplied with the same amount of DMSO as a control. Results are means + standard errors of the means (errors bars) of a triplicate experiment. (C) Northern blot analysis of sigB, sarA, and RNAIII. The transcripts were measured for S. aureus strains grown on TSA plates (48 h) in the absence or presence of 6-TG or MPA as described in panel B. The effect of 6-TG on S. aureus survival in the liver (D) and kidney (E) of tested mice after 5 days of intraperitoneal infection is shown. The limit of detection for organ infection was 100 CFU/organ. 6-TG was dissolved in 0.1 M NaOH, followed by neutralization with HCl. The same amount of NaOH/HCl was used as a control. The statistical difference was determined by using a Student's two-tailed t test.

Microarray analysis of purA and purH mutants in late-stationary-phase cells.

Enhanced pigmentation was observed for pur mutant strains when bacteria were grown to stationary phase (Fig. 3; see also Fig. S2 in the supplemental material). To further study the roles of purine biosynthesis in starvation survival, we performed microarray analysis of transcriptional profiling of both the purA mutant and purH mutant in late-stationary-phase cells and compared the results with those in the wild-type Newman strain. From the microarray data we identified 399 genes with increased transcript levels (≥2.5-fold) and 8 genes with decreased transcript levels in the wild type versus the purA mutant (see Table S2 in the supplemental material). These 407 genes represent ∼16% of the total of annotated genes in the S. aureus Newman genome. Grouping these genes according to their annotated function shows that they belong to several functional categories, primarily energy production and conversion, posttranslational modification, and transcription regulation (see Table S2). Interestingly, citZ, citG, SAV2365, qoxB, hemL, and SAV1117 exhibit decreased expression in the purA mutant compared to expression in the wild-type Newman strain in our microarray analysis. Considering that disrupting these genes led to enhanced pigment production (Fig. 1 and Table 1), we suspect that the decreased expression of these genes might contribute to the enhanced pigmentation observed in the purA mutant when it is grown to late stationary phase (Fig. 3D; see also Fig. S2).

We also compared gene expression in the wild-type Newman strain with expression in the purH mutant strain in late-stationary-phase cells (48 h). We identified 353 genes with increased transcript levels (≥2.5-fold) (see Table S2 in the supplemental material) and 12 genes with decreased transcript levels in the wild-type strain versus the purH mutant (see Table S2). About 195 genes out of 399 that are downregulated in the purA mutant are also downregulated in the purH mutant (see Table S2). In addition, cluster analysis indicates that about 400 (98%) genes that displayed expression differences between the wild-type Newman and purA mutant strains also showed similar expression profiles in the purH mutant and Newman strain (Fig. 6 A).

FIG. 6.

Cluster analysis and Northern blot analysis. (A) Cluster analysis showing that ∼400 genes affected by a purA mutation also show similar expression profiles in a purH mutant compared with those in wild-type Newman. The ratios are log₂ transformed. The scale of gene activities is represented from green (downregulated) to red (upregulated). (B) Northern blot analysis of eight genes which show different expression levels between wild-type Newman and purA, purH, and guaB mutants.

To confirm our microarray results, we performed Northern blot analysis of eight genes (qoxB, sigB, codY, citZ, lexA, clpX, norA, and saeS). The expression profiles of these eight genes as revealed by Northern blot analysis are consistent with the results derived from the microarray analysis (Fig. 6B; see also Table S2 in the supplemental material). Interestingly, the transcripts of these eight genes are upregulated in a guaB mutant (guaB participates in guanine biosynthesis) compared to expression in the wild-type Newman strain (Fig. 6B). This result raises the possibility that the main phenotypic effect of the purine biosynthetic pathway in S. aureus is mediated primarily through purA or adenine biosynthesis. Further studies are required to elucidate the relative contributions from each pathway.

Mutation of either purA or purH led to mostly downregulation of genes in the S. aureus genome, perhaps suggesting a signal for nutrient limitation. Specifically, the expression levels of 54 genes were severely reduced (>6-fold) in both the purA mutant and purH mutant when bacteria were in late stationary phase (see Table S2 in the supplemental material). These genes encode proteins with diverse biological functions, which demonstrated the profound effects of purine biosynthesis on the physiology of S. aureus. Notably, genes involved in energy metabolism are severely repressed in a purA mutant. This includes genes involved in oxidative phosphorylation (qoxB, qoxA, and ctaB), the TCA cycle (sucA and sucB), and β-oxidation (SA0211-SA0215). Expression levels of genes encoding functional components in protein turnover (SA2563, clpX, and SA1270) are also dramatically repressed in a purA or purH mutant. In addition, many regulatory elements are affected in purine biosynthesis mutants. Expression levels of 49 genes encoding proteins with potential regulatory functions (transcriptional regulators and kinases) are affected by purA or purH mutation (see Table S2). Significantly, 14 out of these 49 genes, such as rsbUVW-sigB, sarA, sarR, sarZ, rot, saeS-saeR, codY, tcaR, srrA, and ccpA, are the well-known genes involved in virulence gene regulation in S. aureus.

Disrupting of the TCA cycle and oxidative phosphorylation affect the expression of virulence determinants.

As described above, purine biosynthesis affects virulence gene regulation in S. aureus. To further examine the possibility that the TCA cycle and oxidative phosphorylation may influence pigment production as well as the regulation of other virulence genes, the mRNA levels of global virulence regulators sigB, sarA, and RNAIII were measured in citZ, citG, and SAV2365 mutants. Northern blot analysis revealed that significantly decreased levels of sigB and sarA mRNAs were detected in a citG mutant (Fig. 7 A). In addition, inactivation of citZ, citG, or SAV2365 gave rise to overproduction of RNAIII (Fig. 7A). Transformation of a citZ mutant with the complementing plasmid pYJ335::citZ restored the wild type levels of RNAIII and sigB (Fig. 7B). We further employed semiquantitative plate assays to examine the production and secretion of hemolysin in these strains. As expected, citZ, citG, and SAV2365 mutants exhibit higher hemolytic activity than the wild-type strain (Fig. 7C). In addition, disrupting the sigB gene also led to enhanced hemolytic activity (Fig. 7C).

FIG. 7.

Analysis of representative S. aureus pigment mutants. (A) The transcripts of sigB, sarA, and RNAIII were measured for S. aureus strains grown on TSA plates at 37°C for 24 h. (B) Northern blot analysis of the transcripts of sigB and RNAIII. S. aureus strains were grown on TSA plates for 24 h before total RNA was harvested. (C and D) Hemolytic activity assay. Bacterial strains were inoculated onto plates with a toothpick and incubated at 37°C for 36 h.

Both CtaA and QoxB are functional components of oxidative phosphorylation. We performed hemolysin activity assays on both mutants using TSA agar plates containing 5% sheep blood. Disrupting ctaA generated a strain with reduced hemolytic activity, consistent with a previous report (10). Inactivation of qoxB also resulted in reduced hemolytic activity (Fig. 7D). Complementation of the qoxB mutant with the pKOR1::qoxB plasmid partially restored hemolytic activity to the normal level (Fig. 7D). Northern blot analysis indicates that there is no significant change in sigB, sarA, and RNAIII mRNA levels in a qoxB mutant compared to levels in wild-type strain when bacteria are grown on TSA plates (24 h) despite the increased pigment production and decreased hemolytic activity observed in the qoxB mutant (see Fig. S2C in the supplemental material).

DISCUSSION

Previous studies have revealed that the rsbUVW-sigB system and crtOPQMN operon are required for the production of S. aureus pigment (3, 17, 26, 38, 39, 49). We show here that golden pigment production of S. aureus is also affected by metabolic pathways such as purine biosynthesis, the TCA cycle, and oxidative phosphorylation (Table 1; Fig. 1). Mutations in these pathways lead to enhanced pigmentation of the pathogen, a result that has not been reported in the past. We have also demonstrated the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival of S. aureus and pathogenesis of infection (Fig. 2).

Inactivation of purine biosynthetic genes (purN, purH, purD, or purA) or inhibition of purine biosynthesis led to enhanced pigmentation; this is likely mediated by enhanced expression of sigB (Fig. 3, 4, and 5). In addition, the decreased expression of citZ, citG, SAV2365, qoxB, hemL, and SAV1117 (Fig. 6; see also Table S2 in the supplemental material) might contribute to the enhanced pigmentation observed in pur mutants when bacteria are grown to late stationary phase since disruption of these genes led to enhanced pigment production (Table 1).

While negatively modulating the expression of sigB when bacteria are grown on TSA plates (Fig. 3), purA seems to positively impact the expression of sigB when bacteria are grown to late stationary phase in TSB medium (Fig. 4 and 6). Indeed, the effect of purA on the expression of sigB is pleiotropic and is likely growth phase dependent (Fig. 4). These results are consistent with the notion that the interplay between metabolism and production of virulence factors depends on a large array of factors (46). It is well known that medium constituents (41), growth conditions (16, 48, 50), and stage of growth (Fig. 4) (5, 9, 31) all influence the expression of regulators in S. aureus. Additional work is needed to elucidate underlying mechanisms; however, our data do show a profound effect of purine biosynthesis on the expression of global regulators such as sigB, sarA, and RNAIII in S. aureus.

Disrupting either oxidative phosphorylation or purine biosynthesis leads to a reduction in bacterial in vivo survival, with the largest survival defect observed for the strains impaired for purine biosynthesis (Fig. 2). The increased pigmentation benefits bacteria for survival in certain environments (29, 30). However, disrupting purine biosynthesis may starve the bacteria of essential replicative precursors that also play an important role in S. aureus to escape host immune responses (47). In addition, intact purine biosynthesis is required for full proteolytic activity of S. aureus (Fig. 3). Moreover, gene expression profile analysis indicates that purA or purH is required for full expression of genes that are involved in a wide variety of cellular functions, including energy metabolism, regulation, stress response, and virulence, when S. aureus is grown to late stationary phase (see Table S2 in the supplemental material). Inactivation of some of these genes, for instance, qoxB (Fig. 5C), SAV0862 (1), clpx (15), clp (15), sodA (24), srtA (22), ftsH (28), srrA (42), saeR-saeS (27), sarZ (7), sarA (4), sigB (21), rsbU (21), sucB (35), and dnaD (11), has been shown to result in attenuated virulence of S. aureus in vivo. Thus, the dramatic virulence attenuation of purA and purH mutant strains is most likely due to a combination of several effects, including nutrient limitation, energy metabolism, stress response, and altered global expression of bacterial virulence factors. It has been shown that reactive oxygen species (ROS)-mediated DNA damage is responsible for the attenuated virulence phenotype of Salmonella enterica serovar Typhimurium purine auxotrophs in mice (34). In the current study, the expression of 23 genes involved in DNA repair is also affected by purA or purH mutation (see Table S1).

The disruption of purA can be complemented by adenine in vitro (Fig. 3) but not in vivo (see Fig. S4 in the supplemental material). A similar phenomenon has also been observed for adenine auxotrophs of Bacillus anthracis, and it was proposed that adenine production might be important for host invasion of the bacterium (19). Further experiments are required to test the hypothesis. We further showed that targeting purine biosynthesis with 6-TG resulted in enhanced bacterial killing in vivo, as evidenced by lower bacterial counts in the livers and kidneys of tested mice (Fig. 5). Since significant differences exist between purine biosynthetic pathways in Gram-positive bacteria such as S. aureus and humans (23), optimized inhibitors that shut down purine biosynthesis in bacteria may offer an alternative therapeutic paradigm for treating S. aureus infections.

Inactivation of TCA cycle genes (citZ, citG, and SAV2365) led to enhanced pigmentation (Table 1) while no elevated level of sigB or crtM was observed (Fig. 7A; also see Fig. S2 in the supplemental material). Disrupting these genes yields mutant strains with enhanced hemolysin activity, a phenotype also observed in a sigB mutant (Fig. 7C). In addition, enhanced expression of sigB resulted in decreased hemolysin activity (25). Thus, the enhanced pigmentation of the TCA cycle mutant strains is less likely a result of enhanced activity of SigB. The golden pigment of S. aureus is synthesized from acetyl coenzyme A (acetyl-CoA) and acetoacetyl-CoA via the mevalonate pathway. It is tempting to propose that inactivation of the TCA cycle may direct acetyl-CoA flux to the mevalonate pathway, which could result in increased pigment biosynthesis. In Staphylococcus epidermidis, inactivation of the TCA cycle alters metabolic status, resulting in a redirection of carbon flow from growth into polysaccharide intercellular adhesin biosynthesis (44).

Disrupting ctaA and qoxB gives elevated levels of pigmentation (Fig. 1) and reduced hemolytic activity (Fig. 7). An elevated crtM mRNA level (see Fig. S2A in the supplemental material) was observed in the ctaA mutant. However, Northern blot analysis indicates that there is no significant change in crtM, sigB, and RNAIII mRNA levels in a qoxB mutant compared to levels in the wild-type strain when bacteria were grown on TSA plates (24 h) (see Fig. S2C). In Bacillus subtilis, the qoxB knockout causes a severe reduction of TCA cycle fluxes and an increased overflow metabolism (51). The function of qoxB in S. aureus awaits further investigation. In addition, disrupting either ctaA or qoxB leads to reduction of bacterial survival in vivo although enhanced pigmentation was observed for a ctaA mutant or qoxB mutant. These results further support the notions that the pathogenesis of staphylococcal infections is multifactorial (46) and that the reduced hemolytic activity observed for a ctaA mutant or qoxB mutant may contribute to the attenuated virulence.

Results from this work also suggest a link between metabolic processes and virulence gene expression in S. aureus. This network may play a pivotal role in the extraordinary flexibility of staphylococci to adapt to and cope with changing host environments. The TCA cycle has been proposed to act as a signal transduction pathway, which translates external environmental cues into intracellular metabolic signals in order to modulate the activity of transcriptional regulators in S. epidermidis (44). It has also been reported that the course of staphylococcal infections in mice is likewise dependent upon the nutritional state of the animals at the time of exposure to the infective dose (45). On the other hand, S. aureus cells grown in milk or milk whey in vitro show increased virulence in a mouse model (32). Thus, metabolic processes in both host and S. aureus may be crucial determinants of disease outcome. The findings reported here further emphasize the need for the study of metabolite requirements in the context of infection. We do not yet fully understand the impact of the altered expression levels of virulence regulators and virulence factors in the purine mutants. We speculate that the dramatic virulence reduction of these mutants is due to a combination of multiple factors, including nutrient limitation, energy metabolism, stress response, and altered global expression of bacterial virulence factors. Potent and bacterium-selective small molecules that inhibit purine biosynthesis may lead to the development of a new generation of anti-S. aureus agents.