Transcriptional Profiling of Target of RNAIII-Activating Protein, a Master Regulator of Staphylococcal Virulence
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感染与免疫杂志 2005年第10期
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
Department of Biomedical Sciences Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
ABSTRACT
Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.
INTRODUCTION
Staphylococcus aureus is a gram-positive bacterium that is part of the normal flora of the skin, but it can become pathogenic and cause fatal diseases once it forms a biofilm and/or produces toxins (18, 25). Biofilm formation and toxin production are regulated by a quorum-sensing mechanism, where molecules produced and secreted by the bacteria (autoinducers) reach a threshold concentration and activate signal transduction pathways, leading to activation of the genes that encode virulence factors (22, 27, 32, 35, 37).
To date, two staphylococcal quorum-sensing systems (SQS) have been described. SQS 1 consists of the autoinducer RNAIII-activating protein (RAP) and its target molecule, target of RNAIII-activating protein (TRAP) (4, 5, 20, 21). SQS 2 consists of the molecules encoded by agr (28, 29). The bacteria secrete RAP, a 33-kDa protein, as they multiply (23); when RAP reaches a threshold concentration (in the mid-exponential phase of growth), RAP induces the histidine phosphorylation of its target molecule TRAP (5, 20). The phosphorylation of TRAP leads, in an as-yet-unknown mechanism, to the synthesis of SQS 2, which is composed of the products of the agr system. agr encodes two divergently transcribed transcripts, RNAII and RNAIII (28, 29). RNAII encodes AgrA, AgrC, AgrD, and AgrB, where AgrD is a propeptide that yields an autoinducing peptide (AIP) that is processed and secreted with the aid of AgrB. Once agr is activated and AIP is secreted, AIP induces the phosphorylation of its receptor AgrC, leading to the production of the regulatory RNA molecule termed RNAIII (28). RNAIII upregulates the production of numerous secreted toxins (28). SQS 1 and SQS 2 interact with one another because once AIP is made in the mid-exponential phase of growth, it indirectly downregulates the phosphorylation of TRAP (5). The interplay between the phosphorylation of TRAP and AgrC by their respective autoinducers, RAP or AIP, regulates the expression of adhesion molecules or toxins
Like typical sensors of classical two component systems, TRAP is histidine phosphorylated in the presence of RAP (5, 20); immunoelectron microscopy and Western blotting studies indicate that it is membrane associated (N. Balaban, unpublished data). However, unlike classical sensors, TRAP does not contain a kinase or a transmembrane domain. In addition, TRAP is phosphorylated on three conserved histidine residues and not just one (20). It is therefore suggested that TRAP is a nonclassical signal transducer and that it may be bound to the membrane through other proteins.
TRAP has been demonstrated to be a key molecule regulating pathogenesis because when TRAP expression is inhibited (by mutagenesis) or when TRAP phosphorylation is suppressed (by mutagenesis or by inhibitory peptides), bacteria do not form a biofilm, do not produce toxins, and do not cause disease (1-4, 6, 9-12, 15-17, 19, 20, 33, 36). Here, we show that the expression and phosphorylation of TRAP are necessary for the expression of multiple genes, many of which are virulence factors or their regulators.
MATERIALS AND METHODS
Bacterial strains and culture conditions. S. aureus strains used in this study are the 8325-4 parent strain TRAP+, which is hemolytic; the TRAP– strain, which is nonhemolytic (8325-4 containing a disrupted traP gene and referred to as TRAP); and mutant strain H66A, which is nonphosphorylated and nonhemolytic (8325-4 containing an in-frame mutation in the traP gene, resulting in the exchange of the conserved histidine [His-66] residue with alanine) (20). Strains were grown overnight in 10 ml of tryptic soy broth at 37°C with shaking. Mutant strains were grown also with 100 μg/ml kanamycin. Overnight cultures were used to inoculate (1:100 dilution) 5 ml of fresh tryptic soy broth medium with no antibiotics. Cultures were incubated with shaking at 37°C, and aliquots were removed at the indicated times. It is of note that mutants and parent strains have been shown to have similar growth curves (20).
Microarray. GeneChip S. aureus Genome Array (Affymetrix) contains sequences of S. aureus N315, Mu50, NCTC 8325, and COL. The array contains probe sets to >3,300 S. aureus open reading frames and probes to study >4,800 intergenic region sequences.
RNA isolation and cDNA labeling. Cells were collected by centrifugation, and total RNA was isolated by using the RNeasy Protect (QIAGEN) protocol with some modifications, as follows. At the indicated times, S. aureus bacterial cultures were collected and immediately mixed in 2 volumes of RNA Protect for 5 min at room temperature. Cells were harvested by centrifugation at 5,000 x g for 10 min at 4°C. Cells were lysed by using 100 μl of lysostaphin (3 mg/ml [Sigma-Aldrich]) in TE (100 mM Tris, pH 7.2; 1 mM EDTA) for 10 min at room temperature. RLT buffer (a buffer supplied with the Qiagen kit and supplemented with mercaptoethanol and ethanol) was immediately added. The lysate was then placed on a spin column to bind total RNA. The column was washed multiple times with supplied buffer, and RNA was eluted with diethylpyrocarbonate-treated water. RNA concentration and purity were determined spectrophotometrically. Purified RNA was DNase I treated and RNAsin (RNase inhibitor; Promega). The following was carried out by Genome Explorations, Memphis, TN. cDNA was prepared by using random primers. The RNA-primer mixture (10 μg of RNA and 150 ng of primers) was incubated at 70°C for 10 min, followed by a snap freeze in a dry ice-ethanol bath for 30 s. The sample was centrifuged for 1 min. A deoxynucleoside triphosphate mixture was made (10 mM) and mixed in a final 0.5 mM concentration to the reverse transcription reaction mixture (dithiothreitol and SUPERase·In RNase Inhibitor and SuperScript II [Invitrogen Life Technologies]) was added to the denatured, cooled RNA-primer mixture. A negative control was also included, in which no reverse transcriptase was added; this sample underwent all subsequent sample preparation, hybridization, and analysis steps. The reaction mixture was incubated at 25°C for 10 min, at 37°C for 60 min, and at 42°C for 40 min. The reverse transcriptase was inactivated at 70°C for 10 min and then chilled on ice. RNA was removed by the addition of NaOH and incubation at 65°C for 30 min, followed by neutralization with HCl. cDNA was fragmented by DNase (1 U of DNase I/μg of cDNA) at 37°C for 20 min, DNase was inactivated at 98°C for 10 min, and the fragmented cDNA was applied to the terminal labeling reaction mixture. 3'-Termini labeling was carried out by using the Enzo Bioarray Terminal labeling kit with biotin-ddUTP as described by the manufacturer, with Terminal DNA Transferase. A gel shift assay was used to determine the efficiency of the labeling, which should be >90%. For this assay, labeled material was incubated with avidin prior to electrophoresis, according to the manufacturer's instructions (Enzo) and gel stained with SYBR Gold.
Oligonucleotide array hybridization and analysis (carried out by Genome Explorations). The cRNA pellet was resuspended in 10 μl of RNase-free H2O, and 10.0 μg was fragmented by heat- and ion-mediated hydrolysis at 95°C for 35 min in 200 mM Tris-acetate (pH 8.1), 500 mM potassium acetate, and 150 mM magnesium acetate. The fragmented cRNA was hybridized for 16 h at 45°C to a GeneChip S. aureus Genome Array (Affymetrix). Arrays were washed at 25°C with 6x SSPE (0.9 M NaCl, 60 mM NaH2PO4, and 6 mM EDTA plus 0.01% Tween 20), followed by a stringent wash at 50°C with 100 mM morpholineethanesulfonic acid, 0.1 M Na+, and 0.01% Tween 20. The arrays were then stained with phycoerythrin-conjugated streptavidin (Molecular Probes), and fluorescence intensities were determined using a laser confocal scanner (Hewlett-Packard). The scanned images were analyzed with Microarray software (Affymetrix). Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to constant target intensity (250) for all arrays used. Data analysis was conducted with Microarray Suite 5.0 (Affymetrix), following user guidelines. The signal intensity for each gene was calculated as the average intensity difference, represented by [(PM – MM)/(number of probe pairs)], where PM and MM indicate perfect match and mismatch probes.
To normalize for global systematic variations that could be caused by inconsistencies in loading, each average difference value was divided by the median average difference for a given GeneChip. To identify genes that are below the detection limit of the system, the signal strengths indicative of genes with profiles at the level of noise were determined for each strain as the average signal strength of genes considered absent (via GeneChip algorithms) plus 2 standard deviations.
Northern blot analysis. Early exponential cells were grown from an optical density at 600 nm (OD600) of 0.03 for 6 h (to a postexponential phase of growth) with shaking at 37°C. Cells (200 μl) were collected by centrifugation (2 min at 12,000 x g) and resuspended in 20 μl of lysostaphin in TES buffer (100 μg/ml lysostaphin [Sigma-Aldrich] in 100 mM Tris [pH 7.2], 1 mM EDTA, and 20% sucrose) and incubated for 10 min at room temperature. A total of 20 μl of 2% sodium dodecyl sulfate containing proteinase K (100 μg/ml) was added and vigorously vortexed for 1 min, followed by 10-min incubation at room temperature. The sample was frozen and thawed twice. A 15-μl RNA sample was mixed with 11% deionized glyoxal, 16 mM phosphate buffer, pH 7.0, and 55% dimethyl sulfoxide (final concentrations) and incubated for 1 h at 65°C. RNA loading buffer (Ambion) was added, and the sample was applied to a 1% agarose gel in 10 mM phosphate buffer, pH 7.0, supplemented with 5 mM iodoacetic acid (Sigma-Aldrich). The gel was Northern blotted by dry transfer. Membranes were prehybridized with Rapid-Hyb (Amersham Pharmacia Biotech), followed by hybridization with a PCR-radiolabeled probe derived from the target gene (Table 1), using DNA isolated from S. aureus 8325-4 as a template. Membranes were autoradiographed.
Two-dimensional gel electrophoresis. For preparation of cell extracts, cells were grown from OD600 of 0.03 for 6 h (to postexponential phase) with shaking at 37°C. Cells from 50-ml cultures were collected by centrifugation (7,000 x g) for 10 min at 4°C, washed twice with Tris-EDTA buffer (100 mM-10 mM), and then resuspended in Tris-EDTA buffer containing lysostaphin (100 μg/ml [Sigma-Aldrich]) and DNase I (15 U [Ambion]). After incubation for 10 min at 37°C, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was added (final concentration), and the mixture was incubated on ice for 1 h at 4°C. The lysate was then centrifuged for 30 min at 13,000 x g at 4°C (to remove cell debris). The supernatant was collected, and the protein concentration was determined (Bio-Rad). The proteins were precipitated overnight with 10% (wt/vol) trichloroacetic acid at 4°C. The precipitate was harvested by centrifugation (4°C; 13,000 x g for 10 min), washed several times with 96% (wt/vol) ethanol, and dried. The protein extracts were resolved in an appropriate volume of a solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% carrier ampholytes, 2 mM tributylphosphine, and 0.0002% bromophenol blue. Protein samples (each, 500 μg) were separated on preparative two-dimensional gels with immobilized pH gradient strips (Bio-Rad) in the pH range from 4 to 7. Gels were stained with Coomassie blue G-250 (Bio-Rad). Protein spots were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry at the Maiman Institute for Proteome Research at Tel-Aviv University. Dual-channel images were produced with Delta2D software (Decodon GmbH). The resulting peptide mass fingerprints were analyzed by using the MS-Fit software, GPMAW 4.10, and compared to available genome sequences of S. aureus.
cDNA-PCR. The relative expression levels of spa (protein A), agrC (accessory gene regulator C), rnaIII (-hemolysin), sspA (staphylococcal serine protease; V8 protease), and aur (zinc metalloproteinase aureolysin precursor) genes were determined by cDNA-PCR. As internal standards, the relative expression levels of the epbS gene were used because it was shown by microarray analysis to be equal in the mutant and parent strain. Briefly, cells were grown to the postexponential phase (from OD600 of 0.03 for 6 h) at 37°C. RNA was isolated as described above. DNase-treated RNA was reverse transcribed with SuperScript II as described by the manufacturer (Invitrogen Life Technologies). An equal amount (1/20) of each reaction mixture was then used as a template for PCR amplification. The sequences of the primers are shown in Table 1.
Real-time PCR. Parent S. aureus 8325-4 and TRAP mutant cells were grown to postexponential phase (from OD600 of 0.03 for 6 h) at 37°C. Cells were collected and treated with lysostaphin as described above. RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions, followed by treatment with DNase I (Ambion, Inc.) at 37°C for 20 min according to the manufacturer's instructions. To verify the absence of genomic DNA, PCR was carried out using these DNase I-treated RNA samples as templates, using hld primers. Two micrograms of each RNA sample was used for cDNA synthesis with the ImProm-II Reverse Transcription system, according to the manufacturer's instructions (Promega). Random hexamers (Invitrogen) were used to prime the reaction. A total of 1 μl of the resulting cDNA reaction mixture was used to set up the real-time PCR, using the LightCycler fast-start DNA master SYBR Green I kit (Roche), according to the manufacturer's instructions. The transcripts for hld, hla, clfB, spa, icaR, icaA, sdrC, and sdrD were amplified using the primers shown in Table 1. The gyrB transcripts that are constitutively expressed were used as an internal control. To monitor specificity, the PCR products were analyzed by melting curves and agarose gel electrophoresis. The values are an average of two to three replications normalized with respect to gyrB expression, and the data are expressed as the ratio of cycle threshold (CT) of TRAP/parent 8325-4.
RESULTS
To investigate which genes are regulated by TRAP expression or phosphorylation, TRAP+ S. aureus parent strain 8325-4, the TRAP– mutant, and H66A (a TRAP mutant that acts like a TRAP– mutant because it contains alanine instead of His66) (20) were grown to the postexponential phase (from OD600 of 0.03 for 6 h); cells were collected; and RNA was purified and used for functional genomics experiments (microarray analysis).
Genes upregulated by TRAP. The results presented in Table 2 indicate that multiple genes are upregulated by TRAP. Many of those are virulence factors and their regulatory genes, such as agrABCD (encoding accessory gene regulator ABCD), hld (-hemolysin, which is encoded by RNAIII), hla, hlb, hlgB (-, -, and -hemolysin, respectively), capADFJKL (capsular polysaccharide synthesis enzyme Cap5ADFJKL), lip (triacylglycerol lipase precursor), geh (glycerol ester hydrolase), hysA (hyaluronate lyase precursor), sspA (staphylococcal serine protease [V8 protease]), sspB (cysteine protease precursor), sspC (cysteine protease), SA1725 (staphopain and cysteine proteinase), lrgB (holin-like proteins), plc (1-phosphatidylinositol phosphodiesterase), and aur (zinc metalloproteinase aureolysin precursor) (13, 21, 24-26, 30).
Some of the genes upregulated by TRAP are metabolic: arcA (encoding arginine deaminase), arcB (ornithine transcarbamoylase), arcC (carbamate kinase), ureABC (urease -, -, and -subunits), ureDEFG (urease accessory proteins), pyrR (pyrimidine operon repressor chain A), pyrP (uracil permease), pyrB (aspartate transcarbamoylase chain A), pyrC (dihydroorotase), carA (small-chain carbamoyl-phosphate synthase), and carB (large-chain carbamoyl-phosphate synthase) (7, 13).
Genes downregulated by TRAP. Table 3 shows genes that are downregulated by TRAP. Those include genes involved in cell surface or adhesion molecules such as sdrCD (encoding Ser-Asp-rich fibrinogen-binding, bone sialoprotein-binding proteins), clfB (fibrinogen-binding protein precursor), and spa (immunoglobulin G-binding protein A precursor). Some are involved in adaptive response, such as groEL (encoding a 60-kDa chaperonin), groES (10-kDa heat shock protein), dnaJ (immunoreactive heat shock protein DnaJ), and other genes involved in various cell functions, like pgk (encoding phosphoglycerate kinase 2) (7, 13).
Comparison between genes regulated by TRAP and those shown by other microarray studies to be regulated by agr or sar. Table 4 compares genes regulated by TRAP and those shown by other microarray and real-time PCR studies (7, 8, 13) to be regulated by other virulence regulatory loci like agr or sar. As shown in Table 4, some of the virulence genes regulated by TRAP are also regulated by agr, like hla, hlb, hlgB, capJ, geh, lip, plc, spa, sspA, aur, and hysA. Because TRAP regulates agr, it is likely that TRAP regulates these genes via agr.
Confirmation of microarray data. Some of the microarray results were confirmed by cDNA-PCR, real-time PCR, Northern blotting, and two-dimensional gel electrophoresis. The results in Fig. 1 confirmed by cDNA-PCR that TRAP upregulates agrC, hld (RNAIII), aur, and sspA and downregulates spa while not affecting the expression of the epbS gene. The results shown in Fig. 2 confirmed by real-time PCR that TRAP upregulates hld (RNAIII) and hla, downregulates spa and clfB, and only insignificantly affects icaR and icaA. Real-time PCR results of sdrD and sdrC are contradictory to the microarray results, where according to real-time PCR, less sdrC and sdrD are expressed in the TRAP– mutant (both microarray and real-time tested at least three times).
Figure 3 confirms by Northern blotting that TRAP upregulates hla and hlb.
Figure 4 confirms by two-dimensional gel electrophoresis that NAD-dependent formate dehydrogenase (encoded by fdh) is upregulated, while protein A (spa) and general stress 20U protein (dps) are downregulated by TRAP.
Regulation of genes through TRAP phosphorylation. Transcriptional profiling experiments were carried out using H66A strain that contains an intact TRAP with His 66 replaced by alanine, making that strain nonpathogenic (20). As shown in Tables 2 and 3, similar results were obtained using RNA from postexponential-phase TRAP– cells and H66A cells, suggesting that it is the phosphorylation of TRAP that is important for the regulation of observed genes.
DISCUSSION
Regulation of virulence by TRAP. TRAP has been shown to be an important protein regulating virulence in staphylococci; when TRAP expression or phosphorylation is disrupted by mutagenesis or by inhibitory peptides, no S. aureus- or Staphylococcus epidermidis-induced disease was observed in any of the animal model systems so far tested (1-4, 6, 9-12, 15-17, 19, 20, 33, 36).
As shown here, in the absence of TRAP expression or phosphorylation, multiple virulence factors are not expressed. Those include -, -, -, and -hemolysins; triacylglycerol lipase precursor; glycerol ester hydrolase; hyaluronate lyase precursor; staphylococcal serine protease (V8 protease); cysteine protease precursor; cysteine protease; staphopain-cysteine proteinase; 1-phosphatidylinositol phosphodiesterase; zinc metalloproteinase aureolysin precursor; holing-like proteins; and capsular polysaccharide synthesis enzymes. These proteins have been shown to be important for establishment of the bacteria in the host and subsequent disease progression (13, 21, 24-26, 30).
The data presented here show that TRAP upregulates the agr locus (agrABCD and hld [transcribed by RNAIII]). Most of the virulence factors regulated by TRAP (listed above) have been shown to be regulated by the agr locus (13, 25, 28), and thus we assume that these are regulated by TRAP via agr. No information is available regarding tst, which encodes toxic shock syndrome toxin, as this gene is not present in strain 8325-4. Viewing the extensive list of toxins that are not expressed when the traP gene is disrupted can easily explain why TRAP mutants show no sign of pathogenesis whatsoever in mice, even when injected in very high numbers (20).
Regulation of genes important for biofilm formation. Cells containing TRAP that is defective in expression or phosphorylation adhere less to plastic polymers and to host cells in vitro and do not form a biofilm in vivo (1, 3, 6, 9-12, 15-17). Indeed, in the absence of TRAP expression or phosphorylation, although agr is suppressed as shown here (see below), no substantial upregulation of adhesion molecules is observed. The only virulence genes shown to be upregulated in the absence of TRAP were spa, clfB, and sdr. However, while TRAP-mutants were shown to overexpress protein A (encoded by spa) by every method tested, only in one of four microarray repeats was fibrinogen-binding protein (clfB) shown to be upregulated. This result was, however, confirmed by real-time PCR. The Ser-Asp-rich fibrinogen-binding bone sialoprotein-binding proteins (sdrC and sdrD) were shown by microarray studies to be upregulated in the TRAP– mutant, but opposite results were obtained by real-time PCR. Differences between microarray and real-time PCR results may be due to technical inherent experimental differences, choice of oligonucleotides, sensitivity, or variability in expression levels or detection (31).
Both microarray and real-time PCR data indicate that the icaR gene is marginally downregulated in the TRAP– mutant while not significantly affecting icaA expression.
Other surface proteins, such as the fibronectin-binding protein (encoded by fnbAB), collagen-binding protein (cna), elastin-binding protein (epbS), clumping factor A (clfA), extracellular fibrinogen-binding protein (efb), and extracellular adherence protein (eap) (14, 25) are not overexpressed by the TRAP mutants. Additionally, in the absence of TRAP expression or phosphorylation, the level of expression of genes required for biofilm survival (7) was reduced in arcABC, ureABC, ureDEFG, pyrR, pyrP, pyrB, pyrC, carA, and carB. These results explain our observation that TRAP mutants do not adhere as well as the wild type and do not form a biofilm in vitro or in vivo.
In general, our microarray results are compatible with what was observed with microarray studies using agr mutants (7, 8, 13), suggesting that most genes are regulated by TRAP via agr. The accepted notion has been that phase variation occurs once agr is activated at the mid-exponential phase of growth, where agr downregulates genes encoding adhesion molecules and upregulates genes encoding toxins (25). However, microarray studies using TRAP or agr mutants do not support this hypothesis and show that while multiple genes encoding exotoxins are indeed downregulated if traP or agr is disrupted, most adhesion molecules are not upregulated (7, 13). In addition, in our studies of biofilms, we have shown that under flow conditions, the volume of biofilm is significantly lower by day 4 in TRAP– mutants, while by day 1 the volume of biofilm of TRAP mutant was transiently higher (3), which is also compatible with what was observed with agr mutants (34).
As shown in Table 4, there is some incompatibility in reported regulation of adhesion genes by agr and that of traP. This suggests either that TRAP regulates some of the adhesion genes independently of agr or that there are differences in experimental approaches, use of strains, or use of different gene arrays.
To summarize, the results presented here can easily explain our observation that in the absence of TRAP expression or phosphorylation, the ability of the bacteria to produce toxins, to attach, to form a biofilm, and to survive within the host is seriously compromised; thus in the presence of TRAP inhibitors, staphylococcal diseases are prevented. That disruption of TRAP reduces both biofilm formation and exotoxin production is of major importance when considering TRAP as a target site for therapy, making TRAP a safe therapeutic target site.
ACKNOWLEDGMENTS
We deeply thank Ilya Borovok, Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel, for input and advice. Functional genomics studies were carried out by Genome Explorations, Memphis, TN.
This work was supported by NIH grant R21 AI054858-01 (N.B.).
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Department of Biomedical Sciences Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
ABSTRACT
Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.
INTRODUCTION
Staphylococcus aureus is a gram-positive bacterium that is part of the normal flora of the skin, but it can become pathogenic and cause fatal diseases once it forms a biofilm and/or produces toxins (18, 25). Biofilm formation and toxin production are regulated by a quorum-sensing mechanism, where molecules produced and secreted by the bacteria (autoinducers) reach a threshold concentration and activate signal transduction pathways, leading to activation of the genes that encode virulence factors (22, 27, 32, 35, 37).
To date, two staphylococcal quorum-sensing systems (SQS) have been described. SQS 1 consists of the autoinducer RNAIII-activating protein (RAP) and its target molecule, target of RNAIII-activating protein (TRAP) (4, 5, 20, 21). SQS 2 consists of the molecules encoded by agr (28, 29). The bacteria secrete RAP, a 33-kDa protein, as they multiply (23); when RAP reaches a threshold concentration (in the mid-exponential phase of growth), RAP induces the histidine phosphorylation of its target molecule TRAP (5, 20). The phosphorylation of TRAP leads, in an as-yet-unknown mechanism, to the synthesis of SQS 2, which is composed of the products of the agr system. agr encodes two divergently transcribed transcripts, RNAII and RNAIII (28, 29). RNAII encodes AgrA, AgrC, AgrD, and AgrB, where AgrD is a propeptide that yields an autoinducing peptide (AIP) that is processed and secreted with the aid of AgrB. Once agr is activated and AIP is secreted, AIP induces the phosphorylation of its receptor AgrC, leading to the production of the regulatory RNA molecule termed RNAIII (28). RNAIII upregulates the production of numerous secreted toxins (28). SQS 1 and SQS 2 interact with one another because once AIP is made in the mid-exponential phase of growth, it indirectly downregulates the phosphorylation of TRAP (5). The interplay between the phosphorylation of TRAP and AgrC by their respective autoinducers, RAP or AIP, regulates the expression of adhesion molecules or toxins
Like typical sensors of classical two component systems, TRAP is histidine phosphorylated in the presence of RAP (5, 20); immunoelectron microscopy and Western blotting studies indicate that it is membrane associated (N. Balaban, unpublished data). However, unlike classical sensors, TRAP does not contain a kinase or a transmembrane domain. In addition, TRAP is phosphorylated on three conserved histidine residues and not just one (20). It is therefore suggested that TRAP is a nonclassical signal transducer and that it may be bound to the membrane through other proteins.
TRAP has been demonstrated to be a key molecule regulating pathogenesis because when TRAP expression is inhibited (by mutagenesis) or when TRAP phosphorylation is suppressed (by mutagenesis or by inhibitory peptides), bacteria do not form a biofilm, do not produce toxins, and do not cause disease (1-4, 6, 9-12, 15-17, 19, 20, 33, 36). Here, we show that the expression and phosphorylation of TRAP are necessary for the expression of multiple genes, many of which are virulence factors or their regulators.
MATERIALS AND METHODS
Bacterial strains and culture conditions. S. aureus strains used in this study are the 8325-4 parent strain TRAP+, which is hemolytic; the TRAP– strain, which is nonhemolytic (8325-4 containing a disrupted traP gene and referred to as TRAP); and mutant strain H66A, which is nonphosphorylated and nonhemolytic (8325-4 containing an in-frame mutation in the traP gene, resulting in the exchange of the conserved histidine [His-66] residue with alanine) (20). Strains were grown overnight in 10 ml of tryptic soy broth at 37°C with shaking. Mutant strains were grown also with 100 μg/ml kanamycin. Overnight cultures were used to inoculate (1:100 dilution) 5 ml of fresh tryptic soy broth medium with no antibiotics. Cultures were incubated with shaking at 37°C, and aliquots were removed at the indicated times. It is of note that mutants and parent strains have been shown to have similar growth curves (20).
Microarray. GeneChip S. aureus Genome Array (Affymetrix) contains sequences of S. aureus N315, Mu50, NCTC 8325, and COL. The array contains probe sets to >3,300 S. aureus open reading frames and probes to study >4,800 intergenic region sequences.
RNA isolation and cDNA labeling. Cells were collected by centrifugation, and total RNA was isolated by using the RNeasy Protect (QIAGEN) protocol with some modifications, as follows. At the indicated times, S. aureus bacterial cultures were collected and immediately mixed in 2 volumes of RNA Protect for 5 min at room temperature. Cells were harvested by centrifugation at 5,000 x g for 10 min at 4°C. Cells were lysed by using 100 μl of lysostaphin (3 mg/ml [Sigma-Aldrich]) in TE (100 mM Tris, pH 7.2; 1 mM EDTA) for 10 min at room temperature. RLT buffer (a buffer supplied with the Qiagen kit and supplemented with mercaptoethanol and ethanol) was immediately added. The lysate was then placed on a spin column to bind total RNA. The column was washed multiple times with supplied buffer, and RNA was eluted with diethylpyrocarbonate-treated water. RNA concentration and purity were determined spectrophotometrically. Purified RNA was DNase I treated and RNAsin (RNase inhibitor; Promega). The following was carried out by Genome Explorations, Memphis, TN. cDNA was prepared by using random primers. The RNA-primer mixture (10 μg of RNA and 150 ng of primers) was incubated at 70°C for 10 min, followed by a snap freeze in a dry ice-ethanol bath for 30 s. The sample was centrifuged for 1 min. A deoxynucleoside triphosphate mixture was made (10 mM) and mixed in a final 0.5 mM concentration to the reverse transcription reaction mixture (dithiothreitol and SUPERase·In RNase Inhibitor and SuperScript II [Invitrogen Life Technologies]) was added to the denatured, cooled RNA-primer mixture. A negative control was also included, in which no reverse transcriptase was added; this sample underwent all subsequent sample preparation, hybridization, and analysis steps. The reaction mixture was incubated at 25°C for 10 min, at 37°C for 60 min, and at 42°C for 40 min. The reverse transcriptase was inactivated at 70°C for 10 min and then chilled on ice. RNA was removed by the addition of NaOH and incubation at 65°C for 30 min, followed by neutralization with HCl. cDNA was fragmented by DNase (1 U of DNase I/μg of cDNA) at 37°C for 20 min, DNase was inactivated at 98°C for 10 min, and the fragmented cDNA was applied to the terminal labeling reaction mixture. 3'-Termini labeling was carried out by using the Enzo Bioarray Terminal labeling kit with biotin-ddUTP as described by the manufacturer, with Terminal DNA Transferase. A gel shift assay was used to determine the efficiency of the labeling, which should be >90%. For this assay, labeled material was incubated with avidin prior to electrophoresis, according to the manufacturer's instructions (Enzo) and gel stained with SYBR Gold.
Oligonucleotide array hybridization and analysis (carried out by Genome Explorations). The cRNA pellet was resuspended in 10 μl of RNase-free H2O, and 10.0 μg was fragmented by heat- and ion-mediated hydrolysis at 95°C for 35 min in 200 mM Tris-acetate (pH 8.1), 500 mM potassium acetate, and 150 mM magnesium acetate. The fragmented cRNA was hybridized for 16 h at 45°C to a GeneChip S. aureus Genome Array (Affymetrix). Arrays were washed at 25°C with 6x SSPE (0.9 M NaCl, 60 mM NaH2PO4, and 6 mM EDTA plus 0.01% Tween 20), followed by a stringent wash at 50°C with 100 mM morpholineethanesulfonic acid, 0.1 M Na+, and 0.01% Tween 20. The arrays were then stained with phycoerythrin-conjugated streptavidin (Molecular Probes), and fluorescence intensities were determined using a laser confocal scanner (Hewlett-Packard). The scanned images were analyzed with Microarray software (Affymetrix). Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to constant target intensity (250) for all arrays used. Data analysis was conducted with Microarray Suite 5.0 (Affymetrix), following user guidelines. The signal intensity for each gene was calculated as the average intensity difference, represented by [(PM – MM)/(number of probe pairs)], where PM and MM indicate perfect match and mismatch probes.
To normalize for global systematic variations that could be caused by inconsistencies in loading, each average difference value was divided by the median average difference for a given GeneChip. To identify genes that are below the detection limit of the system, the signal strengths indicative of genes with profiles at the level of noise were determined for each strain as the average signal strength of genes considered absent (via GeneChip algorithms) plus 2 standard deviations.
Northern blot analysis. Early exponential cells were grown from an optical density at 600 nm (OD600) of 0.03 for 6 h (to a postexponential phase of growth) with shaking at 37°C. Cells (200 μl) were collected by centrifugation (2 min at 12,000 x g) and resuspended in 20 μl of lysostaphin in TES buffer (100 μg/ml lysostaphin [Sigma-Aldrich] in 100 mM Tris [pH 7.2], 1 mM EDTA, and 20% sucrose) and incubated for 10 min at room temperature. A total of 20 μl of 2% sodium dodecyl sulfate containing proteinase K (100 μg/ml) was added and vigorously vortexed for 1 min, followed by 10-min incubation at room temperature. The sample was frozen and thawed twice. A 15-μl RNA sample was mixed with 11% deionized glyoxal, 16 mM phosphate buffer, pH 7.0, and 55% dimethyl sulfoxide (final concentrations) and incubated for 1 h at 65°C. RNA loading buffer (Ambion) was added, and the sample was applied to a 1% agarose gel in 10 mM phosphate buffer, pH 7.0, supplemented with 5 mM iodoacetic acid (Sigma-Aldrich). The gel was Northern blotted by dry transfer. Membranes were prehybridized with Rapid-Hyb (Amersham Pharmacia Biotech), followed by hybridization with a PCR-radiolabeled probe derived from the target gene (Table 1), using DNA isolated from S. aureus 8325-4 as a template. Membranes were autoradiographed.
Two-dimensional gel electrophoresis. For preparation of cell extracts, cells were grown from OD600 of 0.03 for 6 h (to postexponential phase) with shaking at 37°C. Cells from 50-ml cultures were collected by centrifugation (7,000 x g) for 10 min at 4°C, washed twice with Tris-EDTA buffer (100 mM-10 mM), and then resuspended in Tris-EDTA buffer containing lysostaphin (100 μg/ml [Sigma-Aldrich]) and DNase I (15 U [Ambion]). After incubation for 10 min at 37°C, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was added (final concentration), and the mixture was incubated on ice for 1 h at 4°C. The lysate was then centrifuged for 30 min at 13,000 x g at 4°C (to remove cell debris). The supernatant was collected, and the protein concentration was determined (Bio-Rad). The proteins were precipitated overnight with 10% (wt/vol) trichloroacetic acid at 4°C. The precipitate was harvested by centrifugation (4°C; 13,000 x g for 10 min), washed several times with 96% (wt/vol) ethanol, and dried. The protein extracts were resolved in an appropriate volume of a solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% carrier ampholytes, 2 mM tributylphosphine, and 0.0002% bromophenol blue. Protein samples (each, 500 μg) were separated on preparative two-dimensional gels with immobilized pH gradient strips (Bio-Rad) in the pH range from 4 to 7. Gels were stained with Coomassie blue G-250 (Bio-Rad). Protein spots were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry at the Maiman Institute for Proteome Research at Tel-Aviv University. Dual-channel images were produced with Delta2D software (Decodon GmbH). The resulting peptide mass fingerprints were analyzed by using the MS-Fit software, GPMAW 4.10, and compared to available genome sequences of S. aureus.
cDNA-PCR. The relative expression levels of spa (protein A), agrC (accessory gene regulator C), rnaIII (-hemolysin), sspA (staphylococcal serine protease; V8 protease), and aur (zinc metalloproteinase aureolysin precursor) genes were determined by cDNA-PCR. As internal standards, the relative expression levels of the epbS gene were used because it was shown by microarray analysis to be equal in the mutant and parent strain. Briefly, cells were grown to the postexponential phase (from OD600 of 0.03 for 6 h) at 37°C. RNA was isolated as described above. DNase-treated RNA was reverse transcribed with SuperScript II as described by the manufacturer (Invitrogen Life Technologies). An equal amount (1/20) of each reaction mixture was then used as a template for PCR amplification. The sequences of the primers are shown in Table 1.
Real-time PCR. Parent S. aureus 8325-4 and TRAP mutant cells were grown to postexponential phase (from OD600 of 0.03 for 6 h) at 37°C. Cells were collected and treated with lysostaphin as described above. RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions, followed by treatment with DNase I (Ambion, Inc.) at 37°C for 20 min according to the manufacturer's instructions. To verify the absence of genomic DNA, PCR was carried out using these DNase I-treated RNA samples as templates, using hld primers. Two micrograms of each RNA sample was used for cDNA synthesis with the ImProm-II Reverse Transcription system, according to the manufacturer's instructions (Promega). Random hexamers (Invitrogen) were used to prime the reaction. A total of 1 μl of the resulting cDNA reaction mixture was used to set up the real-time PCR, using the LightCycler fast-start DNA master SYBR Green I kit (Roche), according to the manufacturer's instructions. The transcripts for hld, hla, clfB, spa, icaR, icaA, sdrC, and sdrD were amplified using the primers shown in Table 1. The gyrB transcripts that are constitutively expressed were used as an internal control. To monitor specificity, the PCR products were analyzed by melting curves and agarose gel electrophoresis. The values are an average of two to three replications normalized with respect to gyrB expression, and the data are expressed as the ratio of cycle threshold (CT) of TRAP/parent 8325-4.
RESULTS
To investigate which genes are regulated by TRAP expression or phosphorylation, TRAP+ S. aureus parent strain 8325-4, the TRAP– mutant, and H66A (a TRAP mutant that acts like a TRAP– mutant because it contains alanine instead of His66) (20) were grown to the postexponential phase (from OD600 of 0.03 for 6 h); cells were collected; and RNA was purified and used for functional genomics experiments (microarray analysis).
Genes upregulated by TRAP. The results presented in Table 2 indicate that multiple genes are upregulated by TRAP. Many of those are virulence factors and their regulatory genes, such as agrABCD (encoding accessory gene regulator ABCD), hld (-hemolysin, which is encoded by RNAIII), hla, hlb, hlgB (-, -, and -hemolysin, respectively), capADFJKL (capsular polysaccharide synthesis enzyme Cap5ADFJKL), lip (triacylglycerol lipase precursor), geh (glycerol ester hydrolase), hysA (hyaluronate lyase precursor), sspA (staphylococcal serine protease [V8 protease]), sspB (cysteine protease precursor), sspC (cysteine protease), SA1725 (staphopain and cysteine proteinase), lrgB (holin-like proteins), plc (1-phosphatidylinositol phosphodiesterase), and aur (zinc metalloproteinase aureolysin precursor) (13, 21, 24-26, 30).
Some of the genes upregulated by TRAP are metabolic: arcA (encoding arginine deaminase), arcB (ornithine transcarbamoylase), arcC (carbamate kinase), ureABC (urease -, -, and -subunits), ureDEFG (urease accessory proteins), pyrR (pyrimidine operon repressor chain A), pyrP (uracil permease), pyrB (aspartate transcarbamoylase chain A), pyrC (dihydroorotase), carA (small-chain carbamoyl-phosphate synthase), and carB (large-chain carbamoyl-phosphate synthase) (7, 13).
Genes downregulated by TRAP. Table 3 shows genes that are downregulated by TRAP. Those include genes involved in cell surface or adhesion molecules such as sdrCD (encoding Ser-Asp-rich fibrinogen-binding, bone sialoprotein-binding proteins), clfB (fibrinogen-binding protein precursor), and spa (immunoglobulin G-binding protein A precursor). Some are involved in adaptive response, such as groEL (encoding a 60-kDa chaperonin), groES (10-kDa heat shock protein), dnaJ (immunoreactive heat shock protein DnaJ), and other genes involved in various cell functions, like pgk (encoding phosphoglycerate kinase 2) (7, 13).
Comparison between genes regulated by TRAP and those shown by other microarray studies to be regulated by agr or sar. Table 4 compares genes regulated by TRAP and those shown by other microarray and real-time PCR studies (7, 8, 13) to be regulated by other virulence regulatory loci like agr or sar. As shown in Table 4, some of the virulence genes regulated by TRAP are also regulated by agr, like hla, hlb, hlgB, capJ, geh, lip, plc, spa, sspA, aur, and hysA. Because TRAP regulates agr, it is likely that TRAP regulates these genes via agr.
Confirmation of microarray data. Some of the microarray results were confirmed by cDNA-PCR, real-time PCR, Northern blotting, and two-dimensional gel electrophoresis. The results in Fig. 1 confirmed by cDNA-PCR that TRAP upregulates agrC, hld (RNAIII), aur, and sspA and downregulates spa while not affecting the expression of the epbS gene. The results shown in Fig. 2 confirmed by real-time PCR that TRAP upregulates hld (RNAIII) and hla, downregulates spa and clfB, and only insignificantly affects icaR and icaA. Real-time PCR results of sdrD and sdrC are contradictory to the microarray results, where according to real-time PCR, less sdrC and sdrD are expressed in the TRAP– mutant (both microarray and real-time tested at least three times).
Figure 3 confirms by Northern blotting that TRAP upregulates hla and hlb.
Figure 4 confirms by two-dimensional gel electrophoresis that NAD-dependent formate dehydrogenase (encoded by fdh) is upregulated, while protein A (spa) and general stress 20U protein (dps) are downregulated by TRAP.
Regulation of genes through TRAP phosphorylation. Transcriptional profiling experiments were carried out using H66A strain that contains an intact TRAP with His 66 replaced by alanine, making that strain nonpathogenic (20). As shown in Tables 2 and 3, similar results were obtained using RNA from postexponential-phase TRAP– cells and H66A cells, suggesting that it is the phosphorylation of TRAP that is important for the regulation of observed genes.
DISCUSSION
Regulation of virulence by TRAP. TRAP has been shown to be an important protein regulating virulence in staphylococci; when TRAP expression or phosphorylation is disrupted by mutagenesis or by inhibitory peptides, no S. aureus- or Staphylococcus epidermidis-induced disease was observed in any of the animal model systems so far tested (1-4, 6, 9-12, 15-17, 19, 20, 33, 36).
As shown here, in the absence of TRAP expression or phosphorylation, multiple virulence factors are not expressed. Those include -, -, -, and -hemolysins; triacylglycerol lipase precursor; glycerol ester hydrolase; hyaluronate lyase precursor; staphylococcal serine protease (V8 protease); cysteine protease precursor; cysteine protease; staphopain-cysteine proteinase; 1-phosphatidylinositol phosphodiesterase; zinc metalloproteinase aureolysin precursor; holing-like proteins; and capsular polysaccharide synthesis enzymes. These proteins have been shown to be important for establishment of the bacteria in the host and subsequent disease progression (13, 21, 24-26, 30).
The data presented here show that TRAP upregulates the agr locus (agrABCD and hld [transcribed by RNAIII]). Most of the virulence factors regulated by TRAP (listed above) have been shown to be regulated by the agr locus (13, 25, 28), and thus we assume that these are regulated by TRAP via agr. No information is available regarding tst, which encodes toxic shock syndrome toxin, as this gene is not present in strain 8325-4. Viewing the extensive list of toxins that are not expressed when the traP gene is disrupted can easily explain why TRAP mutants show no sign of pathogenesis whatsoever in mice, even when injected in very high numbers (20).
Regulation of genes important for biofilm formation. Cells containing TRAP that is defective in expression or phosphorylation adhere less to plastic polymers and to host cells in vitro and do not form a biofilm in vivo (1, 3, 6, 9-12, 15-17). Indeed, in the absence of TRAP expression or phosphorylation, although agr is suppressed as shown here (see below), no substantial upregulation of adhesion molecules is observed. The only virulence genes shown to be upregulated in the absence of TRAP were spa, clfB, and sdr. However, while TRAP-mutants were shown to overexpress protein A (encoded by spa) by every method tested, only in one of four microarray repeats was fibrinogen-binding protein (clfB) shown to be upregulated. This result was, however, confirmed by real-time PCR. The Ser-Asp-rich fibrinogen-binding bone sialoprotein-binding proteins (sdrC and sdrD) were shown by microarray studies to be upregulated in the TRAP– mutant, but opposite results were obtained by real-time PCR. Differences between microarray and real-time PCR results may be due to technical inherent experimental differences, choice of oligonucleotides, sensitivity, or variability in expression levels or detection (31).
Both microarray and real-time PCR data indicate that the icaR gene is marginally downregulated in the TRAP– mutant while not significantly affecting icaA expression.
Other surface proteins, such as the fibronectin-binding protein (encoded by fnbAB), collagen-binding protein (cna), elastin-binding protein (epbS), clumping factor A (clfA), extracellular fibrinogen-binding protein (efb), and extracellular adherence protein (eap) (14, 25) are not overexpressed by the TRAP mutants. Additionally, in the absence of TRAP expression or phosphorylation, the level of expression of genes required for biofilm survival (7) was reduced in arcABC, ureABC, ureDEFG, pyrR, pyrP, pyrB, pyrC, carA, and carB. These results explain our observation that TRAP mutants do not adhere as well as the wild type and do not form a biofilm in vitro or in vivo.
In general, our microarray results are compatible with what was observed with microarray studies using agr mutants (7, 8, 13), suggesting that most genes are regulated by TRAP via agr. The accepted notion has been that phase variation occurs once agr is activated at the mid-exponential phase of growth, where agr downregulates genes encoding adhesion molecules and upregulates genes encoding toxins (25). However, microarray studies using TRAP or agr mutants do not support this hypothesis and show that while multiple genes encoding exotoxins are indeed downregulated if traP or agr is disrupted, most adhesion molecules are not upregulated (7, 13). In addition, in our studies of biofilms, we have shown that under flow conditions, the volume of biofilm is significantly lower by day 4 in TRAP– mutants, while by day 1 the volume of biofilm of TRAP mutant was transiently higher (3), which is also compatible with what was observed with agr mutants (34).
As shown in Table 4, there is some incompatibility in reported regulation of adhesion genes by agr and that of traP. This suggests either that TRAP regulates some of the adhesion genes independently of agr or that there are differences in experimental approaches, use of strains, or use of different gene arrays.
To summarize, the results presented here can easily explain our observation that in the absence of TRAP expression or phosphorylation, the ability of the bacteria to produce toxins, to attach, to form a biofilm, and to survive within the host is seriously compromised; thus in the presence of TRAP inhibitors, staphylococcal diseases are prevented. That disruption of TRAP reduces both biofilm formation and exotoxin production is of major importance when considering TRAP as a target site for therapy, making TRAP a safe therapeutic target site.
ACKNOWLEDGMENTS
We deeply thank Ilya Borovok, Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel, for input and advice. Functional genomics studies were carried out by Genome Explorations, Memphis, TN.
This work was supported by NIH grant R21 AI054858-01 (N.B.).
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