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Journal of Bacteriology, October 2007, p. 7316-7325, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00734-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of IsaA and SceD, Two Putative Lytic Transglycosylases of Staphylococcus aureus

Melanie R. Stapleton,¹ Malcolm J. Horsburgh,² Emma J. Hayhurst,¹ Lynda Wright,¹ Ing-Marie Jonsson,³ Andrej Tarkowski,³ John F. Kokai-Kun,⁴ James J. Mond,⁴ and Simon J. Foster^1*

Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom,¹ Department of Microbiology and Genomics, School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom,² Department of Rheumatology and Inflammation Research, University of Göteborg, Guldhedsgatan 10, S-413 46 Göteborg, Sweden,³ Biosynexus Incorporated, 9119 Gaither Road, Gaithersburg, Maryland 20877⁴

Received 10 May 2007/ Accepted 19 July 2007

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Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and "clumping" of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.

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Staphylococcus aureus is a major cause of skin, soft tissue, respiratory, bone, joint, and endovascular diseases. It can also enter the bloodstream via intravascular access devices, causing bacteremia or septicemia; indeed, infection with S. aureus is a leading cause of nosocomial and community-acquired infections (13, 55, 64). Nasal carriage by patients and care-workers is linked to infection, whereby the organism spreads from the anterior nares to normally sterile parts of the body (61). The emergence of antibiotic-resistant strains, including methicillin-resistant S. aureus, has meant that infections are becoming increasingly difficult to treat (41). Thus, renewed efforts are being made to identify components of this organism which are required for fitness in the host and are expressed during infection, making them potential targets for antimicrobial therapy.

Previously in this laboratory, antigens that are expressed during human infection were identified by screening S. aureus expression libraries with serum samples from patients with confirmed S. aureus bacteremia (6). One of the antigens identified was a putative autolysin, IsaA (immunodominant staphylococcal antigen; SACOL2584) (51). This was in agreement with a previous study identifying IsaA as a major antigen of S. aureus (39). There is a significantly higher titer of immunoglobulin G against IsaA in serum from individuals with confirmed S. aureus disease than in serum from healthy individuals. Furthermore, there is a higher titer of antibodies against IsaA in sera from noncarriers than in sera from nasal carriers (6). Thus, IsaA is expressed in vivo, is antigenic, and so may be a possible candidate for a vaccine against both disease and carriage.

Several autolysins have been found to constitute major surface antigens of S. aureus and S. epidermidis (6, 48). Autolysins hydrolyze specific bonds within the bacterial cell wall peptidoglycan and assist in cell wall expansion, turnover, growth, and cell separation. Previously identified S. aureus autolysins include an N-acetylglucosaminidase which hydrolyzes the ß-1,4 glycosidic linkages between N-acetylglucosamine and N-acetylmuramic acid within the glycan chains of peptidoglycan (Atl), N-acetylmuramyl-L-alanine amidases which cleave the amide bond between N-acetylmuramic acid and the peptide side chain (Atl, Sle1/Aaa), and a Gly-Gly endopeptidase which cleaves the pentaglycine cross-link between peptide side chains (LytM) (15, 22, 29, 45, 49, 63). Lytic transglycosylases are a further class of autolysins, widely distributed among gram-negative bacteria, which cleave the ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues of peptidoglycan, with concomitant formation of 1,6-anhydromuramic acid residues (24). The role of bacterial lytic transglycosylases is largely unknown. They have been proposed to play a role in cell wall turnover and subsequent ß-lactamase induction in Escherichia coli (33), in cell division and induction of the inflammatory immune response via release of peptidoglycan fragments in Neisseria gonorrhoeae (8, 9), and in facilitating the assembly of pili and flagella of Caulobacter crescentus (59). So far, no lytic transglycosylase activity has been demonstrated in S. aureus.

In the present study we analyzed the combined role and regulation of IsaA and SceD in cellular physiology and host-pathogen interactions.

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Bacterial strains, plasmids, and growth conditions. S. aureus and E. coli strains and plasmids are listed in Table 1. Oligonucleotides are listed in Table 2. E. coli was grown in Luria-Bertani (LB) medium using a flask/volume ratio of 1:5 at 37°C with shaking at 250 rpm. Most S. aureus cultures were routinely grown in brain heart infusion (BHI) broth (Oxoid) using a flask/volume ratio of 1:5 at 37°C with shaking at 250 rpm; the exceptions were "aerated" cultures, which were grown using a flask/volume ratio of 1:12.5 with shaking at 250 rpm, and "microaerobic" cultures, which were grown using a flask/volume ratio of 1:2.5 with shaking at 90 rpm. When required, antibiotics or substrates were added to the media at the following concentrations: CdCl₂, 0.25 mM; tetracycline, 5 µg ml^–1; kanamycin, 50 µg ml^–1; neomycin, 50 µg ml^–1; erythromycin, 5 µg ml^–1; lincomycin, 25 µg ml^–1; chloramphenicol, 10 µg ml^–1; and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal), 40 µg ml^–1. Phage transductions using 11 were performed as described elsewhere (44).

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TABLE 1. Bacterial strains and plasmids

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TABLE 2. Oligonucleotides used in this study

Creation of isaA and sceD mutant strains. Allelic replacement of isaA was achieved by amplifying the isaA gene as upstream and downstream fragments using primer pairs OL228/OL229 and OL230/OL231, respectively. The tetracycline resistance gene from pDG1513 was amplified by using primers OL32 and OL33. The three fragments were digested with EcoRI/KpnI, KpnI/BamHI, and KpnI, respectively, and were ligated into EcoRI/BamHI-digested pAZ106. The resulting construct was used to transform S. aureus RN4220 and was resolved via transduction of S. aureus SH1000 with 11.

Allelic replacement of sceD was similarly achieved by amplifying the sceD gene in upstream and downstream fragments by using primer pairs OL243/OL244 and OL245/OL246, respectively, and by amplifying the kanamycin resistance gene from pDG792 by using primers OL252 and OL253. After cloning into pAZ106 as XbaI/KpnI, KpnI/BamHI, and KpnI fragments, respectively, the resulting construct was used to transform S. aureus RN4220 and was resolved via transduction of S. aureus SH1000 using 11. Both the isaA and sceD allelic replacements were confirmed by PCR and Southern blotting.

For complementation of MS001 (isaA), the isaA gene plus a 180-bp sequence upstream of the open reading frame was amplified using primers MS39 and MS40. The fragment was cloned as a HindIII/BamHI fragment into pSK5630, and the resulting construct (pMEL4) and pSK5630 (control) were transformed into MS001, which was followed by selection on agar plates containing chloramphenicol. Successful complementation was confirmed by colony PCR and Western blotting.

Preparation of cell surface proteins. S. aureus strains were grown to log phase and harvested by centrifugation prior to protein extraction as previously described (7).

Overexpression and purification of IsaA and SceD. The isaA and sceD open reading frames were amplified, without the signal sequence, using primer pairs OL238/OL239 and OL250/OL251, respectively, and cloned as NcoI/XhoI fragments into the overexpression vector pET24d+, creating a C-terminal His₆ fusion tag. The resulting plasmids, pMAL48 and pMAL67, respectively, were transformed into E. coli BL21(DE3). The recombinant proteins were expressed as previously described (7). For purification of recombinant SceD, the soluble fraction was passed through a nickel-charged Hi-Trap column (Amersham), and the recombinant protein was eluted using an imidazole gradient. The resulting protein was dialyzed into phosphate-buffered saline (PBS). For purification of recombinant IsaA the insoluble pellet was resuspended in 6 M guanidine-HCl (pH 7.8)-50 mM PBS and passed through a nickel-charged Hi-Trap column. The resulting protein was refolded with the nondetergent sulfobetaine NDSB-201 in the presence of 50 mM HEPES (pH 7.5), as previously described (62). The protein was then dialyzed into 10 mM HEPES (pH 7.5).

SDS-PAGE, zymogram analysis, and Western blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), zymogram analysis, and Western blotting were performed as previously described (14).

Western blot band analysis. The densities of bands were determined using ImageMaster 2D Platinum 6.0 software. Band densities, expressed in arbitrary units, were measured three times, and average values are presented.

Tn551 transposon mutagenesis. Six separate Tn551 transposon libraries were constructed for strains MS027 (SH1000/pRN3208 sceD::lacZ) and MS028 (SH1000/pRN3208 isaA::lacZ), as previously described (53). The insertion frequency was found to be between 81 and 99% for each library.

ß-Galactosidase assays. Log-phase bacteria were used to inoculate fresh media to an optical density at 600 nm (OD₆₀₀) of 0.05; the only exceptions were strains carrying a PspacyycFG fusion, whose cultures were grown to log phase in media containing 10 µM isopropyl-ß-D-thiogalactopyranoside (IPTG), washed three times in BHI medium, and used to inoculate media containing 1 mM or no IPTG. Construction and use of the PspacyycFG strains have been described previously (12). The cultures were then grown for up to 8 h, and samples were removed hourly for analysis. ß-Galactosidase assays using 4-methylumbelliferyl-ß-D-galactopyranoside (MUG) as a substrate were performed as previously described (4).

Viable counting. Samples taken for CFU counting were subjected to low-level ultrasonication (4 µA for 5 min) to disrupt cell clumps. These samples were then serially diluted in PBS and plated onto BHI agar for overnight growth.

Clumping. Strains MS001, SH1000, MS002, and MS003 were grown in 50 ml BHI broth for 6 h, and then the cultures were transferred to tubes on ice and allowed to settle under gravity for 15 min.

Murine septic arthritis pathogenicity model. The murine septic arthritis pathogenicity model used has been described previously (28). Briefly, 10 mice per strain of S. aureus were given an intravenous dose of 4 x 10⁶ CFU. The mice were sacrificed after 13 days, and the kidneys were assessed for staphylococcal persistence.

Nasal colonization studies. Nasal colonization studies were done using the cotton rat model as previously described (31). Briefly, 3 x 10⁸ S. aureus cells were instilled into the noses of 6-week-old cotton rats (five rats per strain). Twenty-one days later the cotton rats were killed, and the numbers of CFU remaining in the noses were determined.

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Mutation of isaA results in an altered cell wall protein profile. Cellular fractionation and SDS-PAGE of an S. aureus SH1000 (wild-type) culture revealed that IsaA is a major protein ionically bound to the cell wall (Fig. 1) and in the culture supernatant (results not shown), as has been shown previously (51). In order to facilitate the study of IsaA, an allelic replacement mutation was generated, creating strain MS001 (isaA), which was verified by Southern blotting (results not shown). The protein profile of MS001 (isaA) demonstrated that at least three proteins were present at visibly increased levels as a result of isaA inactivation (Fig. 1). N-terminal sequencing of two of these proteins identified them as SsaA (staphylococcal secretory antigen A; SACOL2291) and SceD (SACOL2088). SsaA is a homologue of the S. epidermidis protein to which antibodies were detected in patients with infective endocarditis (36), is a member of a group encoded by genes in 10-gene family in S. aureus, and is also called ScaD (48). So far the role of SceD is unknown.

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FIG. 1. SDS (15%, wt/vol)-PAGE analysis of ionically bound cell surface protein extracts of SH1000 and MS001 (isaA). Proteins were identified by N-terminal sequencing after blotting onto a polyvinylidene difluoride membrane and are indicated by arrows; a question mark indicates a protein for which sequence data could not be obtained. The proteins were from the equivalent of 2.0 OD600 units. Lane 1, molecular weight markers (sizes are indicated on the left); lane 2, SH1000; lane 3, MS001 (isaA).

IsaA and SceD demonstrate cell wall hydrolytic activity. Bioinformatic analysis of IsaA (http://cmr.tigr.org/tigr-scripts/cmr/cmrhomepage.cgi; http://www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml) revealed that IsaA and SceD are exclusive paralogues in S. aureus and that they both show sequence similarity to lytic transglycosylase domains (pfam06737) in the C-terminal part of the proteins (Fig. 2A). To investigate this potential function further, SceD and IsaA proteins were overexpressed and purified as recombinant proteins, and the purity was verified by SDS-PAGE (Fig. 2B). Both proteins produced a band of clearing on a renaturing gel containing S. aureus peptidoglycan as the substrate (Fig. 2B).

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FIG. 2. Sequence similarity of IsaA and SceD to E. coli soluble lytic transglycosylase and demonstration of peptidoglycan hydrolase activity. (A) ClustalW alignment of IsaA (SACOL2584) and SceD (SACOL2088) with the lytic transglycosylase domain of E. coli Slt (b4392). Amino acid residue numbers are indicated. Shading indicates (compared to Slt) identical residues (black), conserved substitutions (dark gray), and semiconserved substitutions (light gray). (B) Purified recombinant IsaA and SceD proteins were separated by 13% (wt/vol) SDS-PAGE with 0.1% (wt/vol) S. aureus purified peptidoglycan. Lanes 1, 4, and 5 were stained with Coomassie blue; in lanes 2 and 3 the enzymes were renatured to determine activity. Lane 1, molecular weight markers (sizes are indicated on the left); lanes 2 and 4, 3 µg recombinant IsaA; lanes 3 and 5, 3 µg recombinant SceD. The band for each protein is indicated by an arrow.

Due to the ability of IsaA and SceD to cleave peptidoglycan, the hydrolytic bond specificity of these proteins was investigated. This was attempted both by digestion of purified peptidoglycan with the recombinant proteins and by isolation of peptidoglycan from the wild-type and mutant strains, followed by analysis of the resulting material by high-performance liquid chromatography. However, the results were inconclusive, due to a combination of the low solubility of recombinant IsaA following purification, the low level of activity of the recombinant proteins, and the inability to demonstrate significant levels of lytic transglycosylase products in wild-type S. aureus. Indeed, previous muropeptide and chain length analysis of S. aureus peptidoglycan did not reveal the presence of any anhydromuropeptides (2, 11). This was likely due to their low abundance, as has been shown for Bacillus subtilis, where the proportion of muropeptides containing anhydromuramic acid residues is only 0.4% of the total peptidoglycan material (1). Alternatively, processing of peptidoglycan by lytic transglycosylases may result in release of signature products into the surrounding milieu.

Inactivation of isaA increases transcription of sceD. As the inactivation of isaA leads to elevated levels of the SceD protein in the cell, the effect of IsaA on sceD at the transcriptional level was investigated using fusion strains MS007 (sceD::lacZ) and MS009 (sceD::lacZ isaA), both of which have an intact copy of sceD. The sceD gene was maximally expressed during exponential phase (2 h; 742 MUG units), and inactivation of isaA resulted in an approximately threefold increase in the level of sceD expression (Fig. 3A). Complementation of the isaA mutant via plasmid-borne isaA [MS006 (pMEL4 isaA)] resulted in reduced levels of SceD compared to the levels in the isaA mutant carrying the vector alone [MS005 (pSK5630 isaA)], as judged by Western blotting (Fig. 3B), demonstrating that the effect on SceD was due to isaA inactivation (1.0, 2.0, and 1.5 relative units of band density for MS004 [SH1000/pSK5630], MS005, and MS006, respectively). Interestingly, inactivation of sceD did not result in a corresponding increase in isaA::lacZ expression (strains MS010 [isaA::lacZ isaA⁺] and MS011 [isaA::lacZ sceD]) (Fig. 3C), demonstrating that the regulatory feedback seen when isaA is inactivated does not extend to inactivation of the sceD gene. It is therefore possible that the two proteins have overlapping but distinct roles.

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FIG. 3. Expression of isaA and sceD. (A) Expression of sceD::lacZ reporter fusions in MS007 (SH1000 sceD::lacZ) ( and •) and MS009 (SH1000 sceD::lacZ isaA) ( and ). Filled symbols, OD600 (a representative data set is shown); open symbols, ß-galactosidase activity. The error bars indicate standard errors of the means from three independent experiments. (B) Western blot showing levels of SceD protein in S aureus strains. Ionically bound cell surface proteins from the equivalent of 1.2 OD600 units were separated by 13% (wt/vol) SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and probed with antisera raised against recombinant SceD protein. Lane 1, molecular weight markers (sizes are indicated on the left); lane 2, MS004 (SH1000/pSK5630); lane 3, MS005 (SH1000/pSK5630 isaA); lane 4, MS006 (SH1000/pMEL4 isaA). The bar graph shows relative band densities (in arbitrary units) for the lanes. (C) Expression of isaA::lacZ reporter fusions in MS010 (SH1000 isaA::lacZ) ( and •) and MS011 (SH1000 isaA::lacZ sceD) ( and ). Filled symbols, OD600 nm (a representative data set is shown); open symbols, ß-galactosidase activity. The error bars indicate standard errors of the means from three independent experiments.

In E. coli a link between lytic transglycosylase-generated N-acetylglucosamine-1,6-anhydro-N-acetylmuramic acid tetrapeptide, a major product of cell wall turnover, and induction of ampC expression has clearly been demonstrated (23, 26, 27). It was therefore speculated that the regulatory feedback for sceD expression in strain MS001 (isaA) may result from alterations in secreted cell wall turnover products. However, supernatants from MS001 (isaA) and SH1000 cultures were unable to affect expression of an sceD::lacZ fusion in a wild-type strain (MS007) and an isaA mutant strain (MS009), respectively (results not shown). Similarly, the expression of sceD::lacZ in an isaA mutant strain (MS009) was unaffected by the presence of a wild-type strain (SH1000) in a mixed culture (results not shown). This suggests that any soluble signal does not accumulate in the culture supernatant; however, signaling may still occur. Alternatively, intracellular IsaA may act as a transcriptional regulator, as has recently been shown for toxic shock syndrome toxin (60) and Panton-Valentine leukocidin (35).

Expression of sceD is greatly increased in the presence of NaCl. Various agents and environmental conditions were assessed to determine their effects on sceD and isaA expression in lacZ fusion strains MS007 (sceD::lacZ) and MS010 (isaA::lacZ), respectively; these included paraquat, hydrogen peroxide, a reduced pH level (pH 6), penicillin G, and NaCl (results not shown). The only significant effect was that of 1 M NaCl on sceD expression. This effect was quantified using ß-galactosidase assays with liquid cultures, which revealed that the presence of 1 M NaCl caused an approximately 14-fold increase in the level of sceD expression and that this upregulation was independent of IsaA (Fig. 4A). The effects of NaCl on the levels of SceD protein were confirmed by Western blotting (Fig. 4B) (1.0, 1.4, 1.7, and 1.7 relative units of band density for SH1000, MS001 [isaA], SH1000 with 1 M NaCl, and MS001 [isaA] with 1 M NaCl, respectively).

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FIG. 4. Role of NaCl in the expression of sceD. (A) Expression of sceD::lacZ reporter fusions in MS007 (SH1000 sceD::lacZ) ( and •) and MS009 (SH1000 sceD::lacZ isaA) ( and ). Filled symbols, OD600 (a representative data set is shown); open symbols, ß-galactosidase activity; solid lines, cultures grown in BHI broth; dotted lines, cultures grown in BHI broth with 1 M NaCl. The error bars indicate standard errors of the means from three independent experiments. (B) Western blot showing levels of SceD protein. Lane 1, molecular markers (sizes are indicated on the left); lane 2, SH1000; lane 3, MS001 (SH1000 isaA); lane 4, MS002 (SH1000 sceD); lane 5, SH1000 grown in the presence of 1 M NaCl; lane 6, MS001 (SH1000 isaA) grown in the presence of 1 M NaCl. The bar graph shows relative band densities (in arbitrary units) for the lanes.

Inactivation of sceD results in increased salt sensitivity. As the expression of sceD is induced by the presence of NaCl, the growth of MS001 (isaA), MS002 (sceD), and MS003 (isaA sceD) was assessed in the presence of 2.5 M NaCl. Culture on BHI agar plates showed that there was sceD-dependent impairment of growth in the presence of NaCl, observed as decreased colony size (Fig. 5A and 5B). Results obtained with liquid cultures reflected this, with a trend toward reduced growth for the sceD and isaA sceD mutants compared to the wild-type and isaA mutant strains. The mean yields at 25 h were as follows: wild type, 1.2 x 10⁹ ± 0.1 x 10⁹ CFU/ml; isaA mutant, 1.3 x 10⁹ ± 0.35 x 10⁹ CFU/ml; sceD mutant, 8.0 x 10⁸ ± 2.0 x 10⁸ CFU/ml; and isaA sceD mutant, 7.7 x 10⁸ ± 3.8 x 10⁸ CFU/ml (Fig. 5C). This difference was not seen in cultures grown without addition of NaCl (results not shown). Thus, under stressful conditions SceD is required for normal growth; however, it is not essential as the sceD mutants were still able to grow in media containing 2.5 M NaCl, albeit with lower yields. It is therefore feasible that salt acts simply as a marker of location within the host to increase sceD expression when it is required and/or that SceD is only one of a battery of proteins which facilitate growth in the presence of high salt concentrations. Interestingly, it has previously been shown that growth of S. aureus with 2.5 M NaCl results in increased cell size and altered peptidoglycan architecture (58). Given the elevated levels of SceD in the presence of NaCl, it is possible that this protein has a role in the remodeling of peptidoglycan which facilitates such changes.

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FIG. 5. Growth of isaA and sceD mutants in the presence of 2.5 M NaCl. (A and B) Growth on a BHI agar plate (A) and on a BHI agar plate with 2.5 M NaCl (B). Panel a, SH1000; panel b, MS003 (SH1000 isaA sceD); panel c, MS001 (SH1000 isaA); panel d, MS002 (SH1000 sceD). (C) Growth curves for cultures grown in BHI broth with 2.5 M NaCl. •, SH1000; , MS001 (SH1000 isaA); , MS002 (SH1000 sceD); , MS003 (SH1000 isaA sceD). The values are the means from three independent cultures; the error bars indicate standard errors of the means.

IsaA and SceD affect clumping of S. aureus. Autolysins have been implicated in many cellular functions, including growth, separation, and cell wall turnover (54). Given the ability of recombinant IsaA and SceD to cleave peptidoglycan, these proteins have the potential to affect cell separation. This has already been demonstrated in S. aureus for the bifunctional glucosaminidase/amidase Atl protein (15, 56) and the amidase Sle1 (29). In liquid culture it is possible to visualize defects in cell separation or surface properties as "clumping" of cells. The wild-type and mutant strains were grown in liquid culture, and the degree of clumping was subsequently assessed. MS001 (isaA) showed no clumping, whereas inactivation of sceD (MS002) resulted in a greater degree of clumping than that of the wild-type strain. MS003 (isaA sceD) showed the greatest degree of clumping, with visibly large clusters forming in the culture (Fig. 6A). The cultures were also analyzed by light microscopy and shown to have different degrees of cell separation (Fig. 6B), with the inactivation of sceD resulting in impaired separation. Scanning electron microscopy revealed no obvious differences in the appearance of the individual cells of the wild-type and mutant strains (results not shown). Complementation of the clumping phenotype was attempted in the isaA and sceD mutants. However, the presence of both the complementation plasmid and chloramphenicol in the medium affected cell separation of these strains, thus preventing the assay.

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FIG. 6. Role of IsaA and SceD in cellular clumping. (A) Clumping of cultures grown for 6 h and then allowed to settle under gravity for 15 min. Tube 1, SH1000; tube 2, MS001 (SH1000 isaA); tube 3, MS002 (SH1000 sceD); tube 4, MS003 (SH1000 isaA sceD). (B) Light microscopy (magnification, x400) showing clumping of strains grown for 24 h. Panel a, SH1000; panel b, MS001 (SH1000 isaA); panel c, MS002 (SH1000 sceD); panel d, MS003 (SH1000 isaA sceD).

As IsaA and SceD possess autolytic activity, the apparent increased degree of cell separation evident in the isaA mutant may be explained by the upregulation of SceD. Since isaA inactivation also results in upregulation of SsaA, a protein which has also been demonstrated to possess peptidoglycan hydrolase activity (L. Wright and S. J. Foster, unpublished data), it is possible that it is the concerted activities of both SceD and SsaA which result in this phenotype. However, the exacerbated clumping seen in the sceD and isaA sceD mutants demonstrates that SsaA is unable to compensate for their loss.

Regulation of isaA and sceD expression by known S. aureus transcriptional regulators. To dissect the regulation of isaA and sceD, the lacZ reporter fusions from MS007 (sceD::lacZ), MS008 (sceD::lacZ), and MS010 (isaA::lacZ) were transduced into S. aureus SH1000 strains carrying marked mutations in the known regulatory genes sarA, agrB, saeR, sigB, lytSR, arlRS, rbf, and yycFG (3, 5, 12, 16, 18, 34, 38, 50). Temporal expression of isaA::lacZ and sceD::lacZ was similar in all strains, so changes in ß-galactosidase activity were calculated at the point of maximum expression in each strain. Lesions which did not affect expression are not discussed here.

SarA and YycFG positively regulated isaA expression (2.7- ± 0.5- and 1.6- ± 0.05-fold downregulation of expression, respectively, in the mutant strains [results not shown]). Furthermore, expression assays using aerated and microaerobic cultures indicated that isaA expression is differentially regulated according to oxygen availability (1.6- ± 0.13-fold upregulation in the microaerobic cultures [results not shown]). Interestingly, a recent study showed that IsaA is downregulated under anaerobic conditions, suggesting that control of this gene is complex (17).

With respect to sceD expression, SarA appears to be a negative regulator (2.6- ± 0.5-fold upregulation in the mutant strain), whereas sigma factor B (^B), Agr, and YycFG are positive regulators (1.8- ± 0.1-, 1.6- ± 0.04-, and 1.9- ± 0.03-fold downregulation of expression, respectively, in the mutant strains [results not shown]). This is in accordance with previous studies which showed that levels of SceD are decreased in a ^B mutant (68). However, by far the greatest effect on sceD expression was that of LytSR and SaeR, with approximately 80-fold upregulation in the mutant strains (maxima of 477, 27,661, and 28,824 MUG units for the wild type and lytSR and saeR mutants, respectively) (Fig. 7A). Furthermore, ^B and YycFG are regulators of sceD in the presence of NaCl, as expression was twofold lower in the ^B mutant (maxima of 3,518 and 1,756 MUG units for the wild type and mutant, respectively [results not shown]) and threefold lower in the YycFG mutant (Fig. 7B) under these conditions; note that in order to accommodate the antibiotic resistance of the ^B and YycFG mutational insertions, two different lacZ reporter fusions were used (from MS007 and MS008, respectively), giving rise to different levels of ß-galactosidase activity in the resulting strains. Assays using aerated and microaerobic cultures indicated that in contrast to the expression of isaA, the expression of sceD is not affected by aeration levels (results not shown).

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FIG. 7. Regulation of sceD: expression of sceD::lacZ reporter fusions. Filled symbols, OD600; open symbols, ß-galactosidase activity. (A) Strains MS007 (SH1000 sceD::lacZ) ( and ), MS017 (SH1000 sceD::lacZ lytSR) ( and •), and MS018 (SH1000 sceD::lacZ saeR) ( and ). (B) Strain MS015 (SH1000 sceD::lacZ PspacyycFG) with 1 mM IPTG and no NaCl ( and ), strain MS015 (SH1000 sceD::lacZ PspacyycFG) with 1 mM IPTG and 1 M NaCl ( and •), and strain MS015 (SH1000 sceD::lacZ PspacyycFG) with no IPTG and 1 M NaCl ( and ). The data are a representative data set from three independent experiments that showed less than 20% variability.

The two-component sensor-regulator SaeRS is involved in the regulation of multiple virulence factors required for S. aureus infection (19, 37). The fact that sceD expression is strongly regulated by this system may indicate a role for SceD in the pathogenicity of S. aureus. The finding that both isaA and sceD are regulated by the essential sensor-regulator YycFG is in accordance with the work of Dubrac and Msadek (12), who showed that YycF consensus binding sites are located upstream of the isaA and sceD open reading frames and that recombinant YycF is able to bind to the promoter region of isaA. YycFG is also involved in the regulation of another autolysin, LytM, and in resistance to macrolide-lincosamide-streptogramin B antibiotics (12, 42). It has also been shown that in S. pneumoniae the peptidoglycan hydrolase PcsB is able to complement the essentiality of a YycFG homologue, VicRK, thus demonstrating a vital role for cell wall metabolism in bacterial fitness (43). The inclusion of isaA and sceD in the YycFG regulon demonstrates the potential importance of these proteins in the general fitness and/or virulence of S. aureus.

These findings indicate that the regulation of isaA and sceD gene expression is complex and multifactorial, suggesting that the two proteins are important to S. aureus in many different environments encountered throughout the colonization and infection processes.

Identification of novel regulators of isaA and sceD expression. To identify further regulatory components controlling isaA and sceD expression, Tn551 transposon mutagenesis was carried out in the reporter strains MS008 (sceD::lacZ) and MS010 (isaA::lacZ), and expression was assessed following incubation on agar plates containing X-Gal. Approximately 7,500 colonies were screened from three independent libraries per reporter strain. One regulator which affected isaA expression levels was identified: SrrA (SACOL1535; one clone with a Tn551 insertion at bp 421 downstream of the translational start point). This is which is part of the two-component sensor-regulator SrrAB, which controls gene expression in response to oxygen availability (57, 67). Liquid ß-galactosidase assays revealed that SrrAB is a positive regulator of isaA expression under aerated conditions (2.4- ± 0.04-fold downregulation in the transposon mutant strain [results not shown]). Similarly, only one gene which affected sceD expression was identified, lysA (SACOL1435; four clones with a Tn551 insertion at bp 1067 downstream of the translational start point; 7.4- ± 0.2-fold reduction in expression in the transposon mutant). The lysA gene is the final gene in the lysine biosynthetic operon (66); therefore, the Tn551 insertion effect is most probably due to direct inactivation of lysA rather than polarity. As lysA codes for a lysine biosynthesis protein, diaminopimelate decarboxylase, LysA is unlikely to be a transcriptional regulator per se. However, it has been shown that inactivation of lysA results in reduced activity of ^B (53). Thus, given that both ^B and Agr have the potential to affect sceD expression, it was possible that the effect of the lysA lesion was mediated through one of these regulators. However, sceD expression in the lysA::Tn551 background was not affected by the presence of an agr mutation (results not shown). As a result of the markers used to generate gene insertion mutations and reporter gene fusions, it was not possible to assess directly the requirement for ^B in the lysA-mediated effect on sceD expression. However, Shaw et al. (53) showed that the location of the lysA gene in the S. aureus chromosome represents a large region consisting of approximately 35 kb in which transposon insertion in several of the genes results in similar reduction in ^B activity. One such gene is telA (SACOL1441), which codes for a protein with an unknown function. Interestingly, sceD expression levels were not affected by inactivation of telA (results not shown), suggesting that the mechanism for lysA-mediated regulation of sceD expression may be independent of ^B. It is therefore conceivable that sceD expression is upregulated as a result of changes to the cell wall, for which lysine is a crucial component. Such a basis for gene regulation has been demonstrated in E. coli, where ß-lactamase induction is controlled by the status of cell wall turnover and subsequent recycling (27).

IsaA and SceD are required for virulence. The pathogenesis of strains SH1000 (wild type), MS001 (isaA), and MS0026 (sceD) were assessed in the mouse septic arthritis model of infection (28). By monitoring the bacterial load in the kidneys 13 days after infection, the isaA mutant and the sceD mutant were shown to be slightly attenuated for pathogenicity compared to the wild-type strain, although the differences were not statistically significant (P = 0.064 and P = 0.072, respectively, as determined by a t test) (Fig. 8A). In a separate experiment the pathogenicity of SH1000 was compared with that of MS003 (isaA sceD). This revealed that MS003 is significantly attenuated for virulence in this model (P = 0.019) (Fig. 8B), suggesting that there is a requirement for peptidoglycan remodeling in the pathogenicity of S. aureus.

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FIG. 8. Role of IsaA and SceD in pathogenicity. Mice were intravenously inoculated with 4 x 106 CFU SH1000, MS001 (isaA), MS026 (sceD), or MS003 (isaA sceD), and 13 days later the bacterial loads in the kidneys were assessed. (A) Strains SH1000, MS001 (isaA). and MS026 (sceD). (B) Strains SH1000 and MS003 (isaA sceD). The mean values are indicated by bars.

Another autolysin, Sle1, has also been shown to be involved in S. aureus virulence (29). It is therefore tempting to speculate that aberrant cell separation, due to a lack of specific autolytic activity, might result in impaired dissemination in the host and thus reduced overall fitness.

SceD is required for nasal colonization. Given the salt-dependent regulation of sceD, it is feasible that SceD is most important to S. aureus in the establishment or maintenance of nasal carriage, an environment which presents relatively high salt concentrations (40). We therefore examined the abilities of MS001 (isaA), MS002 (sceD), and MS003 (isaA sceD) to colonize the anterior nares of cotton rats (Fig. 9). The results showed that SceD is essential for nasal colonization in this model (P = 0.018 for strains MS002 and MS003), whereas inactivation of isaA resulted in increased (although not significantly increased [P = 0.054]) colonization. Given the requirement for SceD, the trend towards increased colonization of the isaA mutant is most easily explained by the elevated levels of SceD.

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FIG. 9. Role of IsaA and SceD in nasal colonization. Cotton rats were nasally challenged with 3 x 108 CFU of SH1000, MS001 (isaA), MS002 (sceD), or MS003 (isaA sceD). Twenty-one days later the numbers of CFU remaining in the noses were determined. The mean values are indicated by bars.

Relatively little is known about the S. aureus components required for nasal carriage (46). So far, these components have been primarily surface structures. Wall teichoic acids (synthesized by TagO), ClfB, SrtA, IsdA, KatA, and AhpC have been shown to be involved in nasal colonization (6, 10, 52, 65). Very recently, the surface protein IsdA has also been found to confer resistance to human innate defenses and to be required for survival on human skin, another important site of S. aureus carriage (7a). The requirement for SceD is the first example of a role for an S. aureus autolysin in nasal colonization. As mutations in isaA and sceD do not significantly alter the covalently bound cell wall protein profile of S. aureus (data not shown), it is likely that SceD is directly required for this process. Considering the importance of the cell wall in maintaining cell viability and division, it is likely that alterations to the cell wall architecture are fundamental in the adaptation of S. aureus to different environments. Lytic transglycosylase activity is likely to be necessary to make subtle changes to the peptidoglycan structure required for survival in the various conditions encountered in the host, although the precise nature and location of these changes remain to be elucidated. It is also possible that the ability of cells to separate effectively may be necessary for efficient host interaction.

Concluding remarks. The finding that SceD is essential for nasal colonization raises the possibility that SceD might be effective as a component of a vaccine against carriage of S. aureus. Indeed, it has already been shown that in sera from healthy individuals an elevated titer of antibodies against SceD is associated with noncarriage, albeit not significantly (P = 0.07) (our laboratory, unpublished data). Such a correlation between high-titer antibodies and noncarriage of S. aureus has already been determined for IsdA, which can be used as a vaccine to protect against nasal colonization (6). Furthermore, we have shown that significantly higher titers of immunoglobulin G against SceD are detected in serum from individuals with confirmed S. aureus disease than in serum from healthy individuals (P = 0.003), demonstrating that SceD is expressed during infection and is antigenic (our laboratory, unpublished data). It has also recently been demonstrated that the abundance of SceD is increased in a highly vancomycin-resistant clinical isolate of S. aureus, suggesting a need for altered cell wall structure for resistance to this important antibiotic (47). It is therefore tempting to speculate that SceD plays a sufficiently crucial role in the success of this pathogen to be a potential component of a vaccine against both carriage and disease.

Here we have shown that IsaA and SceD are required for normal growth and for successful host-pathogen interactions of S. aureus, thus reinforcing the link between cell wall metabolism and bacterial fitness in this organism. This study also demonstrated that even subtle alterations to peptidoglycan structure may have significant impacts on cellular physiology and pathogenicity.

 
We thank Tanya Chanturiya (Biosynexus, United States) for technical assistance with the nasal colonization studies and Arthur Moir (University of Sheffield, United Kingdom) for N-terminal protein sequencing. RN4220 PspacyycFG was kindly supplied by Tarek Msadek (Institut Pasteur, France).

This work was funded by Biosynexus Incorporated.

 
* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. Phone: 44 114 222 4411. Fax: 44 114 272 8697. E-mail: s.foster{at}sheffield.ac.uk

Published ahead of print on 3 August 2007.

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Journal of Bacteriology, October 2007, p. 7316-7325, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00734-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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