INTRODUCTION

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Staphylococcus aureus exports a wide variety of exoenzymes, some of which are known virulence factors. Among these enzymes are a variety of proteases, such as metalloprotease, thiolprotease, and the serine proteases V8 and exfoliative toxin A (ETA) and ETB. The best characterized of these, V8 serine protease, was initially isolated from S. aureus strain V8 but has since been shown to be expressed by 67% of the S. aureus isolates tested (2, 11).

Like most exoproteins produced by S. aureus, the Agr regulatory system positively regulates extracellular protease expression in response to increasing cell density (7, 26). In contrast, the Sar regulatory system has a negative effect on protease expression. Recent studies indicate that pleiotropic alterations in exoprotein profiles caused by sar mutations are due to the derepression of staphylococcal proteases, which then leads to exoprotein degradation (7, 9). In addition, McGavin et al. (21) reported that V8 protease modifies the fibronectin-binding phenotype of S. aureus. Thus, secreted proteases could play an important role in the posttranslational regulation of S. aureus exoprotein activity, in addition to modifying host proteins to the benefit of the bacteria.

In this study, we identified a novel S. aureus operon (designated spl) that encodes six previously uncharacterized serine protease-like proteins. This operon is expressed during the transition to stationary phase and is positively regulated by Agr. In addition, two of the Spl proteins expressed by this operon were shown to exhibit proteolytic activity.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. S. aureus strains were routinely grown in tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) containing erythromycin (5 µg/ml), kanamycin (50 µg/ml), or tetracycline (5 µg/ml) where indicated. Escherichia coli strains were grown in Luria-Bertani medium (Fisher Biotech) with ampicillin selection (50 µg/ml). All bacterial cultures were routinely incubated at 37°C and aerated by shaking at 250 rpm.

DNA manipulations. E. coli plasmid DNA was extracted using the Wizard Plus DNA Purification System (Promega, Madison, Wis.). Restriction endonucleases and T4 DNA ligase were purchased from GIBCO-BRL (Gaithersburg, Md.) and used as recommended by the manufacturer. Transformations of E. coli strain DH5 and S. aureus RN4220 were performed using the procedures described by Inoue et al. (17) and Kraemer and Iandolo (19), respectively. S. aureus chromosomal DNA was isolated using the method of Dyer and Iandolo (13), and plasmid DNA was isolated using the Wizard Plus DNA Purification System, except that S. aureus cells were converted to protoplasts using lysostaphin (50 µg/ml). Bacteriophage transductions in S. aureus were performed as described by Shafer and Iandolo (32) using 11 as the transducing phage.

Identification of the spl operon by database analysis. Several proteins with similar physiochemical properties were purified from S. aureus cultures. N-terminal sequence data was used in a BLAST search to screen the S. aureus 8325 (the parent strain of RN6390) sequencing database provided by the Staphylococcus aureus Genome Sequencing Project (University of Oklahoma Health Sciences Center). The N-terminal sequences used originally matched open reading frames found on two separate contigs. One contig contained the splA, splB, and splC genes, while the other contig contained part of splD and all of splE and splF. Since a search of the S. aureus COL genome database (The Institute for Genome Research) indicated that these six genes make up a single operon, the sequence between the two 8325 contigs was PCR amplified and sequenced. The nucleotide sequence of the entire spl operon from 8325 was entered into the GenBank database (accession no. AF271715). The nucleotide sequences of the splB and splC genes were previously identified and included in the GenBank database as orf1 and orf2 (accession numbers U60589 and U63529, respectively).

Purification and N-terminal sequencing. S. aureus strain RN6390 was grown with shaking at 37°C in 5 liters of Todd-Hewitt broth (Difco Laboratories) to late stationary phase. The culture was precipitated in 4 volumes of ethanol for 1 week at 4°C. The precipitate was collected by centrifugation (13,000 × g for 15 min at 4°C) and air dried. Proteins in the pellet were dissolved in 50 ml of water and clarified by centrifugation at 40,000 × g at 4°C for 30 min. After dialysis against multiple changes of water (4°C), the volume of the dialysate was reduced to less than 100 ml by pervaporation. The dialysate was then subjected to flatbed isoelectric focusing (IEF) in a gradient of ampholytes (pH 3 to 10) in Sephadex (Amersham Pharmacia Biotech, Piscataway, N.J.). The IEF gel was divided into 19 fractions. Proteins were eluted from each fraction and then dialyzed exhaustively at 4°C against multiple changes of water. Proteins in each fraction were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and visualized by staining with Coomassie blue. The SplA, SplB, and SplF triplet was further purified using a fast protein liquid chromatography (FPLC) sizing column (Superose 12 HR 10/30; Amersham Pharmacia Biotech). The purified Spl proteins were then separated on an SDS-PAGE gel containing 2.0 M urea and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, Mass.) according to the method of Matsudaira (20). SplB and SplC (from a separate IEF fraction) were further purified by reverse-phase high-pressure liquid chromatography (HPLC) using a Hewlett-Packard HP1090 (Hewlett-Packard, Waldbronn, Germany) apparatus equipped with a 15-cm C₁₈ column (VYDAC, Hesperia, Calif.). Protein was loaded onto the column in 0.1% trifluoroacetic acid and eluted in a gradient of 0 to 60% acetonitrile in 0.1% trifluoroacetic acid with a flow rate of 1 ml/min. The N-terminal amino acid sequences of the purified Spl proteins were determined by Edman degradation (Laboratory for Bioanalysis and Biotechnology, Pullman, Wash.).

Zymographic analysis. S. aureus cultures were grown in 10 ml of TSB to late stationary phase. Cultures then were centrifuged at 6,000 × g for 5 min (4°C), and the supernatant was filter sterilized through a 0.2-µm-pore-size filter (Pall Corporation, Ann Arbor, Mich.). The exoproteins were concentrated by passage through a Centricon 3 centrifugal filter device (Millipore) at 4°C by following the manufacturer's instructions. Protease activity associated with Spl proteins was detected using zymographic analysis essentially as described by Arvidson et al. (2). A 52.5-µl volume of each sample was mixed with 17.5 µl of 4× sample buffer (without -mercaptoethanol) and denatured for 15 min at room temperature. The samples were loaded on an SDS-12% polyacrylamide gel containing 0.1% -casein (Sigma, St. Louis, Mo.), and electrophoresis was performed with a constant voltage of 120 V using buffer precooled to 4°C. Renaturation of proteases was obtained by incubating the gels in 2.5% (vol/vol) Triton X-100 for 30 min with gentle agitation at room temperature. Gels were then placed in development buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl₂, 0.02% Brij 35) for 10 to 12 h with gentle agitation at 37°C and stained with 0.5% Coomassie blue. Zones of hydrolysis were visualized after destaining in a 40% methanol-10% acetic acid solution.

spl operon mutagenesis. The S. aureus spl operon was deleted in strain RN6390 by allele replacement as follows. Initially, a 480-bp DNA fragment spanning a region 3' to splF was PCR amplified using Taq DNA polymerase (GIBCO-BRL) and the splD3 and splD4 primers (5'-GGTAATAAGCCATCAGGTGAAAGCTCTAGAGG-3' and 5'-GCGAACGTTGTTCTGCAGTAATAGAC-3', respectively; see Fig. 2). The DNA product was then digested with XbaI and PstI (underlined sequences) and ligated into the XbaI and PstI sites of pDG647 downstream of the plasmid-encoded erythromycin resistance (Em^r) cassette. This recombinant plasmid was designated pSR1.

spl operon expression construct. For use in complementation experiments, the spl operon (excluding the promoter region) was PCR amplified using primers splA3 (5'-CATTAACTATAAAAATAAGCTTGGAAGGAGG-3') and splD2 (5'-GTATATTTTGTCAGGATCCGGTGAATGTCTAAG-3') and then cloned into the BamHI and HindIII sites (underlined sequences) of pBluescript II KS. This 5.0-kb DNA fragment was then liberated by digestion with BamHI and SalI, ligated to the corresponding sites within pRN5548, and transformed into S. aureus RN4220. Plasmid DNA from one positive clone was then transferred to various S. aureus strains by electroporation. This plasmid construct, designated pSR7, placed the expression of the spl operon under the control of the constitutively expressed -lactamase promoter.

Northern blot analysis. To examine growth phase-dependent expression, cultures were diluted 1:100 in TSB and grown with aeration at 37°C. At 4, 6, 8, 10, and 12 h after inoculation, 10-ml culture aliquots were mixed with an equal volume of ethanol-acetone solution (1:1, vol/vol) and the cells were stored at 20°C. After sampling was complete, the cell suspensions were centrifuged at 6,000 × g (4°C) for 5 min and the pellets were resuspended in 10 ml of TEN buffer (29). The cells were centrifuged again, and the pellets were resuspended in 1 ml of TEN buffer (containing 2.5 M NaCl). The cells were converted to protoplasts by incubation (37°C, 25 min) in the presence of recombinant lysostaphin (50 µg/ml). RNA was isolated using 4 ml of Trizol (GIBCO-BRL) in accordance with the manufacturer's instructions and resuspended in sterile, dimethylpyrocarbonate-treated water containing 0.5% SDS.

Virulence studies. In an initial study to examine if the spl operon contributes to virulence, 29-day-old male Sprague-Dawley rats were given intraperitoneal injections of S. aureus RN6390, KB600, and KB600(pSR7). The dosages used were 10⁷, 10⁸, and 10⁹ CFU in a 500-µl volume. The overall health and viability of the rats were assessed over a 3-day period. After this time, the rats were sacrificed and their organs were examined by visual inspection and bacterial culturing.

	RESULTS

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Purification and physiochemical characterization of novel exoproteins. Fractionation of proteins in S. aureus RN6390 culture supernatants revealed a comigrating group of three extremely basic and similarly sized proteins (SplA, SplB, and SplF) ranging from 26 to 28 kDa (Fig. 1A). Upon preparative IEF, these proteins migrated to the same position in the pH gradients representing pIs of approximately 9.0. Further purification using FPLC resulted in an apparently pure protein preparation containing only these three Spl proteins (Fig. 1A, lane 4). To identify the proteins, they were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and subjected to N-terminal sequencing. The results of this analysis revealed that the N-terminal sequences of the proteins from the upper, middle, and lower bands were ENNVTKVKDTNIFPYTGVVAF, EKNVKEITDATKEPY, and ENTVKQITNTNVAPYSG, respectively.

View larger version (64K): [in this window] [in a new window]   FIG. 1.   Purification of the Spl proteins. (A) The Spl proteins from S. aureus RN6390 grown to stationary phase were fractionated and then separated by SDS-12% PAGE. Lanes: 1, 10-kDa molecular size markers (Gibco-BRL); 2, exoproteins from an RN6390 culture; 3, partially purified exoproteins after IEF fractionation; 4, FPLC fraction containing purified SplA, SplB, and SplF separated in the presence of 2.0 M urea to enhance the resolution of these proteins; 5, purified SplC; 6, purified SplB. The molecular size standards used were the BenchMark Protein Ladder (Gibco-BRL). (B) Zymographic analysis of purified SplC (lane 2) and SplB (lane 3) using casein as the substrate. The molecular size markers used (lane 1) were the BenchMark Prestained Protein Ladder (Gibco-BRL).

Identification of spl genes in the S. aureus database. Comparison of the N-terminal sequences of the purified proteins to the S. aureus 8325 genome database indicated that these proteins were encoded by genes within a six-gene operon (Fig. 2). The N-terminal sequences of the proteins from the upper, middle, and lower bands (Fig. 1A, lane 4) were identical to the deduced amino acid sequences of the first, second, and sixth gene products, respectively. Interestingly, products of all six genes share 43.9 to 94.6% sequence identity with each other and, as a group, contain significant sequence similarity to known serine proteases (Fig. 3 and 4) such as S. aureus V8 protease and the epidermolytic toxins. Notably, each gene product contains the conserved amino acids His-74, Asp-113, and Ser-189 (splA gene product numbering), which make up the classic catalytic triad of trypsin-like serine proteases (Fig. 3). Unlike V8 protease, the Spl proteins appear to be synthesized without propeptides. Because of their sequence similarities to serine proteases, the genes encoding these proteins were designated splA, splB, splC, splD, splE, and splF for serine protease like.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Schematic representation of the S. aureus spl operon (middle) and its disruption by plasmid insertion (top) and allele replacement (bottom). The PCR primers (splA1, splA2, splD3, and splD4) used to generate DNA fragments for allele replacement are indicated. The arrows 5' and 3' to the spl operon represent the putative position of the transcription start site and a putative factor-independent transcription terminator, respectively.

View larger version (79K): [in this window] [in a new window]   FIG. 3.   Sequence alignment of SplA to F with staphylococcal V8 serine protease. High sequence conservation throughout the proteins is evident. Identical residues in all five sequences are boxed. The asterisks indicate residues comprising the catalytic triad.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Sequence identities among the Spl proteins and V8 protease. Shown are the percentages of identical amino acids calculated using paired alignments of the proteins.

Northern analysis. Since the expression of most staphylococcal exoprotein genes is activated during the transition into stationary phase and is dependent on the virulence factor regulator Agr, we examined the expression of the spl operon during various stages of growth. RNA was isolated from S. aureus RN6390 cultures in 2-h increments beginning in exponential phase (4 h) and extending to stationary phase (12 h). RNA was analyzed by dot blot hybridization using an splB-specific DNA probe. As shown in Fig. 5, maximal expression of splB-specific transcripts was detected at the 8-h time point, which corresponded to early stationary phase (unpublished results).

View larger version (90K): [in this window] [in a new window]   FIG. 5.   Temporal regulation of the spl operon. Dot blot analysis of total cellular RNA isolated from RN6390 at 4, 6, 8, 10, and 12 h postinoculation and probed with an splB-specific probe. The 8-h time point corresponds to the transition into stationary phase.

View larger version (82K): [in this window] [in a new window]   FIG. 6.   Northern blot analysis of the spl operon. DNA probes specific for splA, splB, splC, and splF were hybridized with RNAs isolated from RN6390 (lanes 1) and KB601 (lanes 2). Transcript sizes were determined based on the migration of a 0.24 to 9.5-kb RNA ladder (Gibco-BRL).

View larger version (63K): [in this window] [in a new window]   FIG. 7.   Transcriptional regulation of the spl operon. A DNA probe specific for splB was hybridized with RNAs isolated from RN6390 (lane 1), KB600 (lane 2), ALC136 (lane 3), ALC135 (lane 4), and RN6911 (lane 5). Transcript sizes were determined based on the migration of a 0.24 to 9.5-kb RNA ladder (Gibco-BRL).

PCR analysis of clinical isolates. To examine if the spl genes are present in other strains, 11 randomly selected S. aureus isolates (Table 1) were analyzed by PCR amplification using splA- and splD-specific primers. PCR amplification of DNA from these isolates using spl operon-specific primers successfully amplified a 2.8-kb DNA fragment (presumably containing splA, splB, splC, and part of splD) in seven (64%) of the isolates tested (data not shown). With the exception of the Brittingham strain, the results of the PCR survey were confirmed by Southern blot analysis. Although the Brittingham strain produced no DNA fragment in PCR amplification using primers specific to the spl operon, Southern blot analysis revealed the presence of sequences that hybridized to splC- and splD/F-specific probes, but not to probes specific for splA and splB (data not shown).

Virulence studies. The role of virulence factors during the course of infection is most easily determined by generating mutant strains and then assessing their ability to cause disease in an animal model of infection. Accordingly, the entire spl operon was replaced in RN6390 with an Em^r determinant (see Materials and Methods), resulting in strain KB600. SDS-PAGE analysis of the exoproteins produced by this strain confirmed that it failed to produce the Spl proteins (unpublished data). Furthermore, transformation of KB600 with the spl operon expression plasmid pSR7 resulted in restoration of spl expression. To assess the effects of the spl operon on staphylococcal pathogenesis, three dosages (10⁷, 10⁸, and 10⁹ CFU) of RN6390, KB600, and KB600(pSR7) were intraperitoneally injected into 29-day-old rats. Examination of these animals revealed no differences in the 50% lethal dose or the time required to cause death. Furthermore, no differences in the lesions produced by these strains were observed. Thus, the spl operon plays no obvious role in virulence as determined by this animal infection model.

	DISCUSSION

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The results presented here demonstrate the presence of a novel S. aureus six-gene operon (designated spl) that encodes protease activity. Members of the serine protease family, including V8 protease and the ETs, contain a well-conserved active site, known as a catalytic triad (4, 10, 11). This motif is characterized by the presence of a highly reactive serine residue within hydrogen-bonding distance of an imidazole ring nitrogen of histidine and a transition state-stabilizing aspartate residue (6, 14). Similarly, the Spl proteins contain the catalytic triad and exhibit significant sequence similarity to V8 protease and the ETs. Although only the SplB and SplC proteins were purified to homogeneity and shown by zymographic analysis to exhibit protease activity (Fig. 1B), we speculate that all of the Spl proteins are proteases.

The splC gene product was identified previously using antisera from S. aureus endocarditis patients to screen an expression library for genes that are expressed in vivo (28). Analysis of the deduced amino acid sequence of this antigen (designated ORF2) also demonstrated significant sequence similarity to the serine proteases V8 and the ETs. Furthermore, the presence of a catalytic triad within ORF2 was also noted by these authors, leading them to speculate that ORF2 is a protease. The fact that patients' sera reacted with ORF2 indicates that this protein is expressed in S. aureus endocarditis and thus may be an important virulence factor during the course of this infection. In an initial assessment of virulence, a murine intraperitoneal injection model was used in this study to compare the effects of S. aureus RN6390 and its isogenic spl mutant. Although we were unable to observe effects on virulence in this model, more extensive studies using animals more sensitive to S. aureus are ongoing.

It has been presumed that various staphylococcal proteases might function as virulence factors through a variety of mechanisms, including inactivation of antimicrobial peptides, cleavage of human immunoglobin molecules, and allowing dissemination by tissue destruction (15, 31). V8 protease can cleave and inactivate the heavy chains of all human immunoglobin classes in vitro (1, 23) and modify the fibronectin-binding phenotype of S. aureus (21). The degradation of adhesins in the postexponential growth phase could allow the bacteria to spread from initial sites of infection (21). Metalloprotease is required for cleavage of V8 protease from an inactive precursor to a mature protease (12), and the ability of metalloprotease to activate prothrombin in human plasma suggests a role in the disseminated intravascular coagulation that sometimes occurs after systemic infection (34). ETA and ETB, the etiological agents of staphylococcal scalded-skin syndrome, exhibit 25 to 30% similarity to V8 protease. Like V8 protease, the ETs belong to the serine protease family (10) but are dissimilar in their high substrate specificity (25). Epidermolytic activity is lost in mutant toxins with alterations in the catalytic triad region, suggesting that proteolytic activity is essential (27). Recently, Rago et al. (25) revealed that -melanocyte-stimulating hormone is a substrate of ETA and that both ETA and ETB cleave -melanocyte-stimulating hormone. These revelations provide important new insight into the mechanisms of the skin desquamation that is associated with these toxins. Whether the spl gene products have a specific substrate like the ETs or whether they are nonspecific proteases involved in tissue destruction is currently under investigation in our laboratories.