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The Capsular Polysaccharide of Staphylococcus aureus IsAttached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Familyof Enzymes*

Received for publication, March 25, 2014, and in revised form, April 17, 2014 Published, JBC Papers in Press, April 21, 2014, DOI 10.1074/jbc.M114.567669

Yvonne Gar-Yun Chan‡1, Hwan Keun Kim‡, Olaf Schneewind‡§, and Dominique Missiakas‡§2

From the ‡Department of Microbiology, University of Chicago, Chicago, Illinois 60637 and the §Howard Taylor Ricketts Laboratory,Argonne National Laboratory, Argonne, Illinois 60439

Background: Staphylococcus aureus LcpABC attach wall teichoic acids (WTA) to peptidoglycan.Results: S. aureus capsular polysaccharide (CP5) is linked to peptidoglycan in a manner requiring lcpABC genes.Conclusion: Unlike WTA, CP5 attachment is mediated preferentially by LcpC.Significance: LCP proteins display substrate preferences for the transfer of undecaprenyl-bound polymers to peptidoglycan.

Envelope biogenesis in bacteria involves synthesis of interme-diates that are tethered to the lipid carrier undecaprenol-phos-phate. LytR-CpsA-Psr (LCP) enzymes have been proposed tocatalyze the transfer of undecaprenol-linked intermediates ontothe C6-hydroxyl of MurNAc in peptidoglycan, thereby promot-ing attachment of wall teichoic acid (WTA) in bacilli and staph-ylococci and capsular polysaccharides (CPS) in streptococci. S.aureus encodes three lcp enzymes, and a variant lacking all threegenes (�lcp) releases WTA from the bacterial envelope and dis-plays a growth defect. Here, we report that the type 5 capsularpolysaccharide (CP5) of Staphylococcus aureus Newman iscovalently attached to the glycan strands of peptidoglycan. Cellwall attachment of CP5 is abrogated in the �lcp variant, a defectthat is best complemented via expression of lcpC in trans. CP5synthesis and peptidoglycan attachment are not impaired in thetagO mutant, suggesting that CP5 synthesis does not involvethe GlcNAc-ManNAc linkage unit of WTA and may instead uti-lize another Wzy-type ligase to assemble undecaprenyl-phos-phate intermediates. Thus, LCP enzymes of S. aureus are pro-miscuous enzymes that attach secondary cell wall polymers withdiscrete linkage units to peptidoglycan.

Bacteria elaborate capsules as a defense mechanism and pro-tective layer to withstand changes in the environment (1, 2).During infection, encapsulation provides for escape fromopsonophagocytosis and complement-mediated killing (3–5).Capsules are typically composed of high-molecular weightpolysaccharides (CPS)3 that are firmly attached to the cell sur-

face (2, 6, 7). CPS consists of repeat units that vary greatlybetween species as well as within bacterial species, yielding dis-tinct serotypes. Such structural diversity favors escape fromadaptive immune responses. Typically, genes are required forCPS synthesis cluster in defined loci carrying conserved andvariable genes, the latter of which account for serotype speci-ficity. For example, Streptococcus agalactiae may synthesize upto 10 different serotypes (8, 9), and no fewer than 80 and 93capsular serotypes have been reported for Escherichia coli andStreptococcus pneumoniae, respectively (1, 10). Despite theirextensive structural diversity, many capsular polysaccharidesare synthesized via the Wzy-dependent pathway: repeat unitsare assembled onto undecaprenyl-P in the cytoplasm, flippedacross the membrane, and polymerized in a non-processivemanner whereby a dedicated glycosyltransferase transfers anascent capsule polymer from its undecaprenyl-PP carrier tothe non-reducing end of an incoming undecaprenyl-PP-linkedrepeat unit (reviewed in Refs. 1, 2, and 11). This polymerizationmechanism was first elucidated as one of three pathways (Wzy-,synthase-, and ABC transporter-dependent pathways) respon-sible for the synthesis of O-polysaccharide that defines theO-antigen serological specificity in Gram-negative bacteria(11–13). O-Polysaccharide is transferred from its undecapre-nyl-PP carrier onto the lipid A-core at the periplasmic face ofthe plasma membrane (14). Wzy-dependent polymerization isubiquitous in bacteria and based on the genetic conservation ofCPS loci, and appears to account for capsule synthesis in mostGram-positive bacteria (1). Only two synthase-dependent locihave been described including type III CPS of S. pneumoniae(15) and the hyaluronic acid capsule of Streptococcus pyogenes(16). ABC transporter-dependent pathways have not been asso-ciated with capsular polysaccharide synthesis in Gram-positivebacteria.

Like many other pathogens, Staphylococcus aureus elabo-rates a CPS, where up to 13 serotypes have been identified (17,18). The majority of strains isolated from patients and healthyindividuals produce either serotype 5 or 8 (17, 18). The genes

* This work was supported, in whole or in part, by National Institutes of HealthGrant AI38897 from the NIAID and Novartis Vaccines and Diagnostics(Siena, Italy). The authors acknowledge membership within and supportfrom the Region V “Great Lakes” Regional Center of Excellence in Biode-fense and Emerging Infectious Diseases Consortium (GLRCE) supported byNational Institutes of Health NIAID Award 1-U54-AI-057153.

1 Supported by American Heart Association Award 13POST16980091.2 To whom correspondence should be addressed: Dept. of Microbiology, Uni-

versity of Chicago, 920 East 58th Str., Chicago, IL 60637. Tel.: 773-834-8161;Fax: 773-834-8150; E-mail: [email protected].

3 The abbreviations used are: CPS, capsular polysaccharide; LCP, LytR-CpsA-Psr protein; CP5 and CP8, capsular polysaccharides type 5 and type 8 in S.aureus; SrtA, sortase A; L6, ribosomal protein L6; SpA, protein A; TSA, tryptic

soy agar; NAM, N-acetylmuramic acid; WTA, wall teichoic acid; AM, ami-dase; GL, glucosaminidase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 22, pp. 15680 –15690, May 30, 2014Published in the U.S.A.

15680 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014

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specifying CPS in S. aureus are encoded by a 17.5-kb regionwith 16 highly conserved genes, capABCDEFGHIJKLMNOP(97–99% identity between serotypes), and four genes, capHIJK,specifying chemical diversity among serotypes (19, 20). CP5and CP8 share the same repeat unit of three sugar residues, butdiffer in their glycosidic linkages and acetylation (21), becauseof the genetic incongruence of the capHIJK genes betweenstrains. CP5 bears the trisaccharide repeat �(1,4)-D-ManAcA-�(1,4)-L-FucNAc(3OAc)-�(1,3)-D-FucNAc, whereas CP8 iscomprised of �(1,3)-D-ManAcA(4OAc)-�(1,3)-L-FucNAc-�(1,3)-D-FucNAc repeats (22). O-Acetylation of CPS gives riseto antigenic variation and altered immune detection duringinfection and may confer protection against opsonophagocytickilling (21, 23, 24).

The genetic requirement for CP8 synthesis has been describedfor 11 of the 16 genes (25), but only 5 CPS gene products havebeen characterized biochemically (19). Nonetheless, thegenetic composition of the cluster clearly suggests that CPSsynthesis in S. aureus occurs in a Wzy-dependent manner. Inboth S. agalactiae and S. pneumoniae, the biosynthetic enzymesare encoded by an operon beginning with a conserved gene,cpsA/cps2A (1, 26 –28). cpsA deletion mutants of S. pneu-moniae and S. agalactiae produce less capsule, a phenotypeoriginally attributed to a possible role in transcription (29 –31).CpsA encodes a protein that belongs to a family of highlyhomologous proteins, LytR-CpsA-Psr (LCP), present often asnumerous paralogues in most Gram-positive organisms (32). Instreptococcal species, CpsA/Cps2A has recently been impli-cated in mediating the transfer/attachment of capsule from itsundecaprenyl-phosphate lipid carrier onto peptidoglycan,where loss of cpsA, lytR, and psr result in reduced capsule pro-duction, and release of the polymer into culture supernatants(31, 33). A cpsA-like homologue is conspicuously missing in theCPS gene cluster of S. aureus, and tethering of the capsule to theenvelope of staphylococci has not been revealed. In this work,we explore the nature of capsule attachment in staphylococci.We show that the capsule can be extracted along with cell wallfragments and can be released by chemical treatment or enzy-matic degradation of peptidoglycan. Unlike wall teichoic acid(WTA), attachment of CP5 is not dependent on TagO, theenzyme that synthesizes the linker unit tethering WTA to pep-tidoglycan. However, similar to WTA, CPS is not attached tomurein sacculi in a mutant lacking all three lcp genes, �lcp. Weconclude that CP5 is attached to peptidoglycan in a mannerrequiring LCP enzymes.

EXPERIMENTAL PROCEDURES

Growth Media and Reagents—S. aureus strains were propa-gated in tryptic soy broth or agar at 37 °C with antibiotic selec-tion when necessary. Erythromycin and chloramphenicol wereused at 10 �g ml�1, spectinomycin at 200 �g ml�1, and tunica-mycin at 1 �g ml�1, unless specified otherwise. For capsuleproduction, S. aureus strains were cultured on Columbia agarplates supplemented with 2% NaCl and the appropriate antibi-otic. E. coli strains were grown in lysogeny broth supplementedwith ampicillin at 10 �g ml�1 when needed. To assess growth ofstaphylococcal strains, stationary phase cultures were normal-ized to A600 � 3.0, diluted 1:100 into 3 ml of tryptic soy broth,

and incubated with shaking at 37 °C. Sample aliquots weretaken at 3.5 h and serially diluted to determine colony-formingunits (cfu).

Bacterial Strains and Plasmids—The clinical isolate S. aureusNewman (wild-type) was used for this study (34, 35). Newmanvariants carrying a deletion of tagO or lcpB were generated byallelic exchange using plasmid pKOR1 (36) and as described(37, 38). Similarly, Newman lcpA::spec was generated by allelicexchange using plasmid pKOR1 encompassing 1-kb DNAsequences upstream and downstream of the lcpA codingsequence with the intervening spectinomycin resistance cas-sette replacing lcpA. DNA segments upstream and downstreamof lcpA were cloned following PCR amplification of genomicNewman DNA using primers pairs with sequences: upstreamprimer pair 5�-GGGGACAAGTTTGTACAAAAAAGCAGG-CTGTGTCTTTGAACATTTCAACGGTCTATATCG-3� and5�-cacgaacgaaaatcgatATCCATATTTACCTACCTTATATC-TTCAAAAATAG-3�; downstream primer pair 5�-caataaaccc-ttgcataGAAGATTAAAAATAAACAAGGCGATTTCTATC-ATAC-3� and 5�-GGGGACCACTTTGTACAAGAAAGC-TGGGTTCTCGCAAGGGCTGAATTGGCCATAATTTCG-TTGG-3�. The spectinomycin element was amplified with aprimer pair bearing the sequences 5�-GGTAGGTAAATATG-GATatcgattttcgttcgtgaatacatgttat-3�, and 5�-GCCTTGTTTA-TTTTTAATCTTCtatgcaagggtttattgttttctaaaatct-3� from plas-mid pJRS312 (39). Mutant alleles capO::erm, lcpA::erm, andlcpC::erm were transduced with bacteriophage �85 lysatesderived from variants with bursa aurealis insertional lesions(40). The double and triple mutant strains were generated by�85 phage transduction of the marked lcpA::erm or lcpC::ermlesions into the lcpB background to generate lcpAB and lcpBCmutants, and by transducing the lcpA::spec allele into the lcpCand lcpBC backgrounds to generate the lcpAC and lcpABC vari-ants. The triple mutant lcpABC is referred as �lcp. All mutantalleles were verified by DNA sequencing.

Plasmids for the purification of recombinant LcpA, LcpB,and LcpC were generated as follows: the DNA encoding theextracellular portion of the LCP proteins (LcpA-(55–327),LcpB-(29 – 405), and LcpC-(35–315); numbers indicate theamino acid position of the predicted extracellular domain of theproteins) was amplified by PCR from S. aureus Newman chro-mosomal DNA, and DNA fragments were cloned into pET-24bby using the BamHI and XhoI restriction sites. lcpA, lcpB, andlcpCDNA fragments were amplified using primer pairs, 5�-gg-atccgAGCGGTGTAGAATATGCCAAG-3� and 5�-ctcgagATC-TTCATCTAAAAAGTCTTTAATAGC-3�; 5�-NNggatccg-ACGTCCCAAGATGCATTCGAATC-3� and 5�-NNctcgag-ATTTACAACACCATTTTGGTTATTTGAAGC-3�; and5�-NNggatccgGCTAAAATTTTTATTACTGGTAATAA-G-3� and 5�-NNctcgagCTCTAGATTATCTTTTAATAACT-TAGTAC-3�. Cloned genes were verified by sequencing.

Subcellular Fractionation of CPS Producing Staphylococci—Overnight cultures of S. aureus were spread onto Columbiaagar plates and incubated at 37 °C for 16 h. Cells harvested fromone plate were suspended in 5 ml of 50 mM Tris-HCl (pH 7.5)and normalized by optical density at 600 nm (A600 � 50). Oneml of suspension was vortexed vigorously and split into two0.5-ml aliquots. One aliquot was treated with 10 �g ml�1 lyso-

LCP Enzymes Attach Capsule to Staphylococcal Peptidoglycan

MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15681


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staphin at 37 °C for 2 h (total fraction). The second aliquot wascentrifuged for 5 min at 21,000 � g and the sediment containingintact cells was separated from the supernatant. Molecules inthe supernatant fractions were subsequently digested withDNase (50 �g ml�1) and RNase (50 �g ml�1) supplementedwith 2 mM MgCl2 for 2 h at 37 °C, and then treated with PronaseE (1 �g ml�1) at 37 °C for 16 h. Extracts were centrifuged at21,000 � g for 10 min, and soluble material was analyzed for thepresence of CP5.

Preparation of Intact Murein Sacculi—Murein sacculi wereprepared as described (41). Briefly, cells were scraped offColumbia agar, suspended in 4% SDS and boiled for 30 min.Cells were subsequently washed five times in water to removedetergent, and broken in a bead-beating instrument (MP Bio-medicals). Murein sacculi were sedimented by centrifugation(10,000 � g, 10 min), washed twice with water, and suspendedin 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 20 mM MgCl2, fordigestion with DNase (10 �g/ml) and RNase (50 �g/ml) for 2 hat 37 °C, and then treated with trypsin (100 �g/ml) for 16 h at37 °C. Murein sacculi were again sedimented by centrifugation,suspended in 1% SDS, boiled for 15 min to inactivate enzymes,washed twice with water, once with 8 M LiCl, once with 100 mM

EDTA, twice with water, once with acetone, and twice morewith water. Purified murein sacculi were suspended in water,normalized to optical density (A600 � 10), and stored at �20 °Cuntil further use.

Extraction of Macromolecules Bound to Peptidoglycan—CP5that associated with murein sacculi was liberated throughchemical or enzymatic treatment as follows. Briefly, samplescontaining CP5 were incubated for 16 h with 40% hydrofluoricacid at 4 °C, or 0.1 M NaOH at room temperature. The remain-ing insoluble peptidoglycan material was sedimented andwashed three times with 1 M Tris-HCl (pH 7.5), and incubatedwith lysostaphin as described above. For enzymatic release ofCP5 associated with intact murein sacculi, sample aliquots (50�l of A600 � 10) were solubilized by digestion with either lyso-staphin (10 �g) in 0.5 M Tris-HCl (pH 7.5), autolysin amidase(20 �g), or glucosaminidase (20 �g) in 0.1 M Tris-HCl (pH 7.0),at 37 °C for 16 h. Insoluble material was removed by centrifu-gation (21,000 � g, 10 min), and soluble material was trans-ferred to a new tube for analyses.

Immunoblotting—To examine the protein contents ofextracts from murein sacculi preparation or subcellular frac-tionations, sample aliquots were mixed (v/v) with sample buffer(0.1 M Tris-HCl, pH 8.0, 4% SDS, 20% glycerol, 2 mM �-mercap-toethanol, 0.04% bromphenol blue), heated at 90 °C for 10 min,and subjected to 12% SDS-PAGE prior to electrotransfer topoly(vinylidene difluoride) (PVDF) membranes. In someinstances when total bacterial loads were monitored, staphylo-cocci from washed cultures were lysed with lysostaphin andprecipitated with 7% trichloroacetic acid. The precipitates werewashed once with acetone, dried, and solubilized in 100 �l of0.5 M Tris-HCl (pH 8.0), 4% SDS, separated on 12% SDS-PAGEprior to transfer to PVDF membranes. Membranes wereblocked by incubation in 2% bovine serum albumin for 1 h atroom temperature. To prevent binding of primary antibodies toprotein A, 0.8 mg of human IgG was added to 10 ml of blockingbuffer. After incubation (1 h, room temperature), immune

serum (primary polyclonal antibodies at a dilution of 1:5,000 foranti-50 S ribosomal subunit L6, anti-SrtA, and anti-SpA, and ata dilution of 1:15,000 for anti-LcpA, anti-LcpB, and anti-LcpC)was added, and the membranes were incubated for an addi-tional hour. Membranes were washed 3 times for 10 min inTBS-T (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween20), and incubated 1:20,000 in secondary antibody (goat anti-rabbit coupled to IRDye� 680) for 1 h. Membranes were washedagain in TBS-T, and fluorescence at 700 nm was measured withan infrared scanner (Li-Cor Biosciences Odyssey imager).Immunoblotting for CP5 was performed in a similar mannerexcept that samples were spotted directly onto nitrocellulosemembrane (primary polyclonal antibodies were used at a dilu-tion of 1:5,000).

Purification of Proteins—E. coli XL1 Blue was used for thepurification of glutathione S-transferase fusions to the autoly-sin amidase (AM) (42) or glucosaminidase (GL) domains (43).E. coli BL21(DE3) was used for the purification of soluble Lcpproteins carrying an N-terminal His6 tag. Bacterial cultureswere grown to A600 � 0.6 in LB with 100 �g ml�1 of ampicillinat 37 °C and lcp expression was induced with 1 mM isopropyl1-thio-�-D-galactopyranoside overnight at room temperature.Bacterial cells were sedimented by centrifugation (10,000 � g,10 min) and suspended in lysis buffer A (50 mM Tris-HCl (pH7.5), 150 mM NaCl) for the purification of GST fusions and inPBS (pH 7.4), containing 20 mM imidazole for His6-tagged pro-teins. Cells were lysed by passage through the French Pressurecell (twice at 15,000 p.s.i.). Crude lysates were cleared by cen-trifugation (100,000 � g, 30 min), and the supernatant wasloaded by gravity flow onto glutathione-Sepharose beads (GEHealthcare) pre-equililibrated or nickel-nitrilotriacetic acidbeads (Qiagen) pre-equilibrated in their respective lysis buffer.Columns were washed with 20 volumes of lysis buffer, andbound proteins were eluted with either 20 mM reduced gluta-thione or 500 mM imidazole. Proteins in the eluates were dia-lyzed into the lysis buffer, quantified by bicinchoninic acidassay (Pierce), and kept at 4 °C for immediate use or storedfrozen at �80 °C.

Purification and Conjugation of CP5 for Antibody Produc-tion—S. aureus CP5 was purified according to a previously pub-lished method (44) with some modifications. S. aureus Reyn-olds (capsular polysaccharide type 5) was grown on Columbiaagar plates supplemented with 2% sodium chloride to inducecapsular polysaccharide synthesis. After overnight incubationat 37 °C, bacteria were scraped off agar plates and suspended in25 ml of buffer A (50 mM Tris-HCl, 2 mM MgSO4, pH 7.5). Thestaphylococcal suspension was incubated with lysostaphin (150�g/ml) at 37 °C with slow rotation for 2 h. Samples were thentreated with DNase and RNase (50 �g/ml) for an additional 2 hat 37 °C, followed by overnight treatment with Pronase E (5units/ml) at 37 °C. Samples were centrifuged to sediment bac-terial debris and the supernatant was sterilized by filtration(0.2-�m Millipore nitrocellulose filter). The filtrate was dia-lyzed against buffer B (50 mM NaOAc, 10 mM NaCl) and loadedonto a MonoQ anion exchange column equilibrated with bufferB, the recorded absorbance of collected fractions at 206, 215,260, and 280 nm was measured. CPS was eluted with 400 ml oflinearly increasing gradient of salt (0.01–1 M NaCl). Collected




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fractions were analyzed to identify elution of capsular polysac-charide (immunoblotting), peptidoglycan (Morgan Elson reac-tion), and teichoic acid (phosphate content). Fractions contain-ing capsular polysaccharide were pooled and treated with 10mM NaIO4 to oxidize contaminating teichoic acid polymers for30 min at room temperature. Samples were dried in a rotatorylyophilizer. The resulting powder was dissolved in 1 ml of sterilewater and chromatographed on a Sephacryl S-300 columnequilibrated with buffer B. Fractions containing capsular poly-saccharide were pooled and lyophilized. A full wavelength scan(200 – 800 nm, Varian) was performed to determine the purityof isolated CP5. Nuclear magnetic resonance (NMR) spectros-copy was used to verify the structure of the capsular polysac-charide by comparison with previously published spectra (22).For NMR analysis, dried polysaccharide was solubilized intodeuterated water and analyzed using a 500 MHz NMR spec-trometer (Varian Inova 500) at an indicated probe temperatureof 50 °C.

Purified CP5 was chemically conjugated to conventional car-rier protein, CRM197, a non-toxigenic mutant of diphtheriatoxin using the carbodiimide cross-linker. First, CRM197 wasderivatized with adipic acid dihydrazide using 1-ethyl-3(3-di-methyl-aminopropyl)-carbodiimide-HCl in MES buffer (pH6.0). Similarly, lyophilized capsular polysaccharide was acti-vated with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimidein the presence of N-hydroxysulfosuccinimide (sulfo-NHS) inMES buffer (pH 6.0). The coupling reaction was performed atroom temperature for 3 h and cross-linker reactivity wasquenched with 1 M Tris-HCl buffer (pH 8.0). The sample waschromatographed on Superose 12 column equilibrated withPBS. The void volume containing the polysaccharide conjugatewas pooled and dialyzed against PBS.

Generation of Rabbit Polyclonal Antibodies—For antibodygeneration, rabbits (Charles River Laboratories, 6-month-oldfemale New Zealand White rabbits) were immunized with 500�g of CP5-CRM197 conjugate or purified Lcp proteins emulsi-fied in Complete Freund’s adjuvant (Difco). For booster immu-nizations, samples were emulsified in incomplete Freund’sadjuvant and injected 24 or 48 days following the initial immu-nization. Rabbits were bled on day 60 and serum stored forfuture use.

Assessment of the in Vitro Activity of Murein Hydrolases—The enzymatic activity of purified AM and GL derived fromautolysin was assessed by using osmotically stable bacterial cellsas substrates. Briefly, S. aureus Newman (25 ml) grown toA600 � 1 and bacteria were sedimented by centrifugation at8000 � g for 10 min. Bacteria in the pellet were suspended in 5ml of water, 5 ml of ethanol/acetone (1:1) was added, and sam-ples were incubated on ice for 30 min. This treatment renderedthe cells osmotically stable. Cells were washed with 20 ml ofice-cold water and suspended in 100 �l of 0.1 M Tris-HCl (pH7.5) for incubation with lysostaphin, or in 0.1 M Tris-HCl (pH7.0) for incubation with AM or GL. Cell wall-anchored proteinsreleased by these treatments were recovered in the supernatantof acetone-treated protoplasts after centrifugation at 21,000 �g for 10 min. Protein A in the soluble extracts was identified byimmunoblot using a rabbit polyclonal anti-SpA serum (�-SpA)from our laboratory collection.

Transmission Electron Microscopy—Bacteria were washedtwice with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, bathed infixative (2% glutaraldehyde, 4% paraformaldehyde, 0.1 M

sodium cacodylate buffer) overnight at 4 °C, and post-fixedwith 1% OsO4 in 0.1 M sodium cacodylate buffer for 60 min.Fixed samples were stained in 1% uranyl acetate in maleatebuffer for 60 min, serially dehydrated with increasing concen-trations of ethanol, embedded in spurr resin for 48 h at 60 °C,thin sectioned (90 nm) using a Reichert-Jung Ultracut device,and post-stained in uranyl acetate and lead citrate. The sampleswere imaged on the FEI Tecnai F30 with a Gatan charge-cou-pled device (CCD) digital micrograph.

Statistical Analyses—The abundance and release of macro-molecules from the cell wall of wild type, mutant, and comple-mented strains was analyzed for statistical analysis by analysisof variance with Dunnett’s multiple comparison test. All datawere analyzed using Prism (GraphPad Software, Inc.); p values�0.05 were deemed significant.

RESULTS

Purification of CP5 for the Generation of Antibodies—Toexamine the capsule of S. aureus, we first purified CP5 from thewell characterized strain Reynolds and raised a polyclonal anti-body. S. aureus Reynolds was grown on Columbia agar supple-mented with 2% NaCl to favor capsule production (45) andcapsular materials were extracted following sequential incuba-tions of bacterial cells with lysostaphin, DNase and RNase, andPronase E. Soluble materials obtained following these treat-ments were subjected to anion-exchange chromatography, andeluted fractions with the highest absorbances at 206 and 215nm were pooled (Fig. 1A). The presence of CP5 in these frac-tions was later verified by dot blot with specific rabbit CP5antiserum (Fig. 1A, lower panel). The pooled fractions werechromatographed on a Sephacryl S-300 size exclusion column.Fractions with the highest absorbances at 206 and 215 nm andlowest protein content were pooled (Fig. 1B). Enrichment forcapsule was documented retroactively by dot blot (Fig. 1B,lower panel). The purity of CP5 in the pooled fractions wasverified by an absorbance scan (200 – 800 nm) revealing thatprotein contaminants were indeed separated following sizeexclusion chromatography and no longer present in the pooledfractions (Fig. 1C) and its structure was confirmed by NMRspectroscopy (Fig. 1D). The chemical shifts of protons associ-ated with the N- or O-acetyl groups were identified at 2.0 –2.1ppm, and chemical shifts of protons (H) associated with thesugar molecules were matched to 3.5–5.0 (H1–H5) and 1.2–1.3ppm (H6), as previously reported (22). This purified CP5 wasused to generate polyclonal antibodies in a rabbit followingchemical conjugation to the conventional carrier protein,CRM197.

The Capsular Polysaccharide of S. aureus Is Associated withMurein Sacculi—Previous work suggested that extraction ofCPS could be enhanced under acidic conditions or followingincubation of staphylococci with lysostaphin (7, 46, 47). None-theless, routine preparations of CPS involved the recovery ofcapsular materials from the supernatant of previously auto-claved bacterial suspensions (47). Here, we asked whether CP5is covalently attached to the murein sacculi of S. aureus New-




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man and, if so, by what mechanism. Staphylococci were har-vested, boiled in SDS, washed extensively, and broken in a beadbeater. Cellular molecules (nucleic acids and proteins) wereenzymatically degraded, and murein sacculi were purified. Lys-ostaphin was used to hydrolyze peptidoglycan and thus solubi-lize macromolecules otherwise associated with murein sacculi.When spotted on a nitrocellulose membrane and stained with arabbit polyclonal anti-CP5 serum (�-CP5), immunoreactivespecies were observed in murein sacculi of wild type strain S.aureus Newman (WT), but not from the murein sacculi of avariant lacking capO, a gene that is known to be essential forcapsule synthesis (48) (Fig. 2A, left panel). To examine thechemical nature of the capsule linkage to peptidoglycan, wefirst asked whether it was sensitive to hydrolysis with weak acid,a treatment known to release phosphodiester and O-acetylbonds (41, 49, 50). Incubation of murein sacculi with hydroflu-oric acid removed all CP5 immune reactive signals, indicatingthat capsule is linked to peptidoglycan via an acid labile bond(Fig. 2A, left panel). To account for the possibility that hydro-fluoric acid treatment removed O-acetyl moieties, therebyabrogating antibody binding to CP5, we subjected murein sac-culi to alkaline lysis with 0.1 M NaOH for 16 h at room temper-ature, a treatment that removes O-acetyl groups (21, 49). Alka-line lysis did not abolish antibody detection of CP5 (Fig. 2A,right panel), indicating the CP5 linkage to peptidoglycan isindeed susceptible to hydrofluoric acid hydrolysis.

CP5 Association with Murein Sacculi Does Not Require TagO—Attachment of WTA polymers to peptidoglycan requires alinker unit that is synthesized by TagO a member of the WecA

family of proteins that links UDP-GlcNAc and undecaprenylphosphate (C55-P) to generate C55-PP-GlcNAc (51). We won-dered whether S. aureus CP5 synthesis requires tagO expres-sion as well. Murein sacculi were extracted from the S. aureusNewman tagO variant, treated with lysostaphin, and spotted onnitrocellulose membrane. In a complementary approach, wildtype S. aureus Newman was grown in the presence of low con-centrations of tunicamycin, an antibiotic from Streptomycesclavuligerus that inhibits the glycosyl transferase activity ofTagO at 1 �g ml�1. At higher concentrations (10 –20 �g ml�1),tunicamycin inhibits MraY, an enzyme essential for peptidogly-can biosynthesis. Dot blot analysis of murein sacculi digestedwith lysostaphin revealed continued synthesis and associationof CP5 with peptidoglycan regardless of whether the tagO geneand its product were genetically or chemically inactivated (Fig.2, A and B). These data indicate that tagO expression and TagOactivity are dispensable for CP5 synthesis and assembly in thecell wall envelope. Of note, bioinformatics analyses (BLASTsearches) suggest that TagO and MraY are the only members ofthe WecA family present in S. aureus. Interestingly, depletionof WTA, elicited upon tagO deletion or tunicamycin inhibition,resulted in an increase in CP5 as compared with wild type (Fig.2B).

CP5 Is Linked to the Glycan Strands of Peptidoglycan—Thepeptidoglycan of S. aureus is comprised of the repeating disac-charide, N-acetylmuramic acid (NAM)-(�1– 4)-N-acetylgluco-samine. The D-lactyl moiety of N-acetylmuramic acid is amide-linked to tetrapeptides that are cross-linked via pentaglycylcross-bridges to wall peptides of adjacent glycan strands (Fig.3A) (52). Where pentaglycine cross-linking does not occur, theterminal pentaglycyl cross-bridge is linked to surface proteins, areaction that is catalyzed by the transpeptidase sortase A (SrtA)(Fig. 3A) (53, 54). Several murein hydrolases have been charac-terized for the substrate specificities in cleaving the staphylo-coccal peptidoglycan. AM cut the amide bond that links thewall peptide to NAM, thereby liberating glycan strands. GLcleave the N-acetylglucosamine-(�1– 4)-NAM bond, andendopeptidases, such as lysostaphin, cut the pentaglycinecross-bridge (Fig. 3A). Murein sacculi prepared from wild typestrain Newman or the isogenic capO variant were incubatedwith lysostaphin, AM, or GL (43). If CP5 were attached to NAM

FIGURE 1. Purification of staphylococcal type 5 capsular polysaccharide.A, extract derived from S. aureus Reynolds grown on Columbia salt agar weresubjected to anion-exchange chromatography on a MonoQ column equili-brated with buffer B (50 mM NaOAc, 10 mM NaCl), and absorbance of elutedfractions was recorded at 206 (blue) and 215 nm (pink). Type 5 capsular poly-saccharide (CP5) elution was identified by immunoblotting with specificrabbit antiserum. Green bar indicates sample fractions that were pooled forfurther purification. B, capsular polysaccharide sample (A) was chromato-graphed on a Sephacryl S-300 size exclusion column equilibrated with bufferB and absorbance at 206 and 215 nm was recorded. CP5 elution was identifiedby immunoblotting with specific antiserum. The bracket indicates fractionspooled for lyophilization. C, absorbance scan (200 – 800 nm) of purified CP5.D, 500 MHz one-dimensional 1H NMR spectroscopy of purified CP5 at 50 °C.

FIGURE 2. S. aureus CP5 is attached to peptidoglycan in a TagO-indepen-dent manner. A, murein sacculi extracted from Newman wild-type (WT),capO, and tagO strains grown on Columbia agar supplemented with 2% NaClwere incubated for 16 h with () or without (�) 40% hydrofluoric acid (HF) at4 °C, or 0.1 M NaOH at room temperature. Acid- or base-treated sacculi weresubsequently neutralized and digested with lysostaphin. Solubilized materi-als were spotted onto a nitrocellulose membrane and probed for CP5immune reactivity using the �-CP5 antiserum. B, WT, capO, and tagO strainswere grown on Columbia agar supplemented with 2% NaCl in the presence of0, 1, or 5 �g/ml of tunicamycin (Tun), which inhibits TagO-glycosyl transferaseactivity. Murein sacculi were extracted, solubilized by lysostaphin treatment,and analyzed by immunoblotting using the �-CP5 antiserum.




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or N-acetylglucosamine residues of peptidoglycan, incubationwith lysostaphin or AM, which eliminate peptidoglycan cross-linking, would be expected to release capsule from murein sac-culi. In contrast, GL cleavage would not be expected to solubi-lize the capsule, as cut glycan strands would remain associatedthrough a network of cross-linked wall peptides. These predic-tions were tested. As expected, treatment of murein sacculiwith lysostaphin, AM, or GL caused a decrease in turbidity, or adecline in A600 (Fig. 3B). Large amounts of CP5 were releasedfrom murein sacculi treated with lysostaphin or AM (Fig. 3, C

and D). In contrast, incubation with GL did not promote releaseof CP5 from murein sacculi (Fig. 3, C and D).

To ensure that the purified AM and GL displayed theirexpected activities, staphylococci were incubated with eitherlysostaphin for 2 h, or AM or GL for 16 h at 37 °C. Solublematerial was separated from cells by centrifugation, and ana-lyzed by immunoblotting for the presence of protein A (SpA), aSrtA-anchored surface protein (Fig. 3E). Equal amounts of SpAwere released upon treatment with either AM or GL, whereaslysostaphin, as expected (the enzyme cuts the anchoring pointof surface proteins), was more effective (Fig. 3, C and D). Takentogether, these data indicate that CP5 is attached to the glycanstrands of the staphylococcal peptidoglycan.

LCP Enzymes Are Required for CP5 Attachment toPeptidoglycan—We wondered whether S. aureus lcp genes arealso involved in the attachment of CP5 to the staphylococcalenvelope albeit that capsule anchoring requires neither mureinlinkage units nor the expression of tagO. To test this possibility,lcp mutant alleles were generated in S. aureus Newman, asMSSA1112 and its variants (37) do not elaborate CP5. Com-pared with the wild type parent strain Newman, the �lcp vari-ant displayed a 2-log reduction in viability, as shown by enu-merating colony forming units (cfu) in broth cultures grown tothe same A600 (Fig. 4, A and B); this phenotype was alsoobserved with the MSSA1112 parent (37). In comparison, thetagO mutant exhibited only a 1-log reduction in cfu as com-pared with Newman (Fig. 4B). Transmission electron micro-graphs of wild type bacteria revealed regular-shaped cells withsepta placed at the mid-cell (Fig. 4C, top row). Cells of the tagOvariant often failed to separate fully and displayed aberrant sep-tal placement (Fig. 4C), as reported before (55). Micrographs of�lcp cells revealed defects similar and more exacerbated thanthose of the tagO mutant (Fig. 4C). Consistent with previousreports that neither tagO nor �lcp elaborate cell wall-boundteichoic acid, the outer electron dense layer was missing in theenvelope of �lcp variant Newman (Fig. 4C) (37, 55).

To examine the contribution of lcp genes for display of CP5in S. aureus Newman, the wild-type, tagO, capO, LCP single(lcpA, lcpB, lcpC), double (lcpAB, lcpAC, lcpBC), and triplemutant (�lcp) strains, and the �lcp variant complemented withsingle LCP genes (�lcp/plcpA, �lcp/plcpB, �lcp/plcpC) werecultured on Columbia agar with NaCl. Bacteria were scrapedfrom plates, suspended in 50 mM Tris-HCl (pH 7.5) buffer, andvortexed vigorously. Samples were sedimented and non-cell-associated macromolecules were recovered in the supernatant.The presence of CP5 and cytosolic ribosomal protein L6 insupernatant fractions was examined by dot blot and immuno-blot, respectively (Fig. 5, A and B, S fractions). The data indi-cated that staphylococci grown on Columbia agar were notprone to lysis, as ribosomal protein L6 was not detected in thesupernatant fractions. The variant lacking lcpC accumulatedCP5 in the supernatant fraction. The lcpAB mutant did notdisplay a defect in CP5 retention. The lcpAC, lcpBC, and triple(�lcp) mutants displayed levels of CP5 release commensurateto the lcpC mutant (the quantification for three independentexperiments is shown in Fig. 5C). Of note, the increased releaseof capsule in the supernatant fraction could be rectified byexpression of lcpC on a plasmid (plcpC) in the �lcp mutant (Fig.

FIGURE 3. CP5 association with glycan strands of peptidoglycan. A, sche-matic of peptidoglycan structure and site of cleavage by muralytic enzymes.WTA is attached to the 6�-hydroxyl of N-acetylmuramic acid (NAM), whereassortase A substrates such as protein A (SpA) are anchored to the pentaglycinecross-bridge. B, murein sacculi from S. aureus Newman WT and capO strainswere incubated at 37 °C with muralytic enzymes lysostaphin (L), AM, GL, orbuffer (B) alone. Turbidity (A600), as a measure of peptidoglycan cleavage, wasmonitored for 12 h. C, murein sacculi solubilized as in B were spotted ontonitrocellulose membrane and analyzed for CP5 release by immunoblot usingthe �-CP5 antiserum. A representative blot of seven replicates is shown. D,CP5 release from C was quantified by densitometry measurements. Thevalue of 1 was attributed to samples treated with lysostaphin and used fornormalization. Statistical differences compared with buffer treatmentwere determined by analysis of variance with Dunnett’s multiple compar-ison test (***, p � 0.001). E, muralytic enzyme activity of lysostaphin, AM,and GL was assessed by release of the SpA protein (black arrow) from S.aureus Newman. Staphylococci, treated with ethanol/acetone to createosmotically stable cells, were incubated with lysostaphin, AM, GL, orbuffer (B) at 37 °C. Solubilized material was separated from intact cells bycentrifugation, resolved by SDS-PAGE, and analyzed by immunoblottingfor protein A release using �-SpA antibody and for cell lysis using �-L6(ribosomal protein L6) antiserum. Numbers to the left of the blots indicatemolecular mass markers (kDa).




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5, A and C). Plasmid-encoded lcpA and lcpB failed to reducecapsule release in the supernatant fractions of �lcp mutant bac-teria to statistically significant levels (Fig. 5, A and C). To doc-ument that similar numbers of cells were processed for the dotblot analyses, bacterial cultures were lysed with lysostaphin andthe protein content in these total extracts was examined for thepresence of ribosomal protein L6 by immunoblot (Fig. 5B).Next, murein sacculi were extracted from washed bacterial cellspreviously scrapped off Columbia agar plates. Dot blot analysesusing �-CP5 antibodies showed that all strains except for thecapO mutant produced CP5 (data not shown). However, cap-sular polysaccharide was greatly diminished in murein sacculifrom the lcpC mutant and absent from sacculi of the �lcp var-iant (Fig. 5, A and D). Mutants lacking lcpA or lcpB had no effecton CP5 attachment whether deleted individually or in combi-nation. Interestingly, the �lcp defect was restored by overpro-ducing anyone of the three lcp genes on a plasmid (Fig. 5, A andD). Together, these data show that lcp genes are essential for theattachment of CP5 to peptidoglycan. Moreover, lcpC encodesthe key enzyme for immobilization of capsular polysaccharideto the staphylococcal cell wall.

LcpC Requirement for Capsule Attachment Is Not Dueto Preferential Expression over LcpA and LcpB—The datadescribed above suggest a key contribution of LcpC toward CP5attachment in the envelope of staphylococci. Because bacteriaare grown to stationary phase on Columbia agar, a conditionthat is more favorable for capsule production, we wonderedwhether LcpC production might be specifically enhanced.Staphylococcal strain Newman and the isogenic �lcp mutantwere grown either in tryptic soy broth or Columbia agar. Bac-teria were normalized to similar optical densities and lysed withlysostaphin. Proteins in extracts were separated by SDS-PAGEand transferred to PVDF membranes for incubation with poly-clonal antibodies against LcpA, LcpB, and LcpC, as well as SrtA,which served as a loading control (Fig. 6). This analysis did notreveal any noticeable changes in LCP protein levels, indicatingthat lcpC is not differentially expressed compared with lcpAand lcpB when cells are induced for CP5 expression. Thus,LcpC appears to have evolved to preferentially recognize cap-sular polysaccharide synthesis intermediates for attachment tothe cell wall envelope.

DISCUSSION

Using India ink negative staining, Gilbert first reported cap-sule production in S. aureus, studying a strain that had beenisolated from a patient with “acute ulcerative gonococcal endo-carditis” (56). When kept for 1 month on infusion agar slants,Gilbert observed two forms of colonies, smooth (S) forms withoverall morphology and staining pattern similar to the originalisolate and rough (R) forms, variants lacking capsule produc-tion. Gilbert (56) also reported that the supernatant forms weremore virulent when inoculated into the peritoneal cavity ofguinea pigs. A typing scheme was eventually developed basedupon preparation of absorbed rabbit antiserum to prototype S.aureus strains and led to the identification of eight capsularserotypes (46). This method revealed the prevalence of CPStypes 5 and 8 in hospital isolates of S. aureus and assigned theheavily encapsulated strains M and Smith diffuse to serotypes 1and 2 (17). It is now appreciated that CP5 and CP8 are 2 of 13different S. aureus serotypes and account for 80% of humanisolates (reviewed by Ref. 18).

Genes involved in the synthesis of bacterial capsular polysac-charides are organized in large genomic clusters where thepresence of conserved genes, i.e. homologues shared amongmany different capsule clusters, provide insights on the mech-anism of capsule assembly (1, 57). The genetic content of S.aureus CPS clusters suggests that staphylococci employ theWzy-dependent mechanism that involves the formation ofrepeat units and non-processive polymerization for the synthe-sis of capsule (1). A model for this pathway in S. aureus is shownin Fig. 7. The final step of the synthesis pathway involves trans-fer of translocated capsule polymer from its bactoprenol carrieronto peptidoglycan. This step is poorly characterized and themolecular principles for capsule attachment in Gram-positivebacteria have rarely been addressed. For example, NMR analy-sis of mutanolysin cleaved sacculi revealed that CPS type III ofGBS is linked via a glycosidic linker to N-acetylglucosamineresidues of peptidoglycan (6), whereas the Lancefield group Bcarbohydrate of GBS appears to be linked to N-acetylmuramicresidues of peptidoglycan with an intervening linkage unit (6,58). In Bacillus anthracis, the capsule is composed of a poly-D-�-glutamate polymer, and is also attached to peptidoglycan.

FIGURE 4. Growth and morphology of S. aureus NM strains. S. aureus Newman strains WT, tagO, and �lcp were assessed for growth. Overnight cultures werenormalized to A600 � 3.0, diluted 1:100, and monitored by recording optical density over time (A) and colony forming units in 10-fold serial dilutions of theculture after a 3.5-h incubation of cultures at 37 °C (B). C, transmission electron micrographs of Newman WT, tagO, and �lcp strains after a 3.5-h incubation ofcultures at 37 °C. Far right panels show magnification depicting the cell envelope. Black arrowheads denote outer electron-dense WTA layer; gray arrowheadspoint to the absence of this layer.




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However, precursor polyglutamate filaments are not synthe-sized on undecaprenyl-phosphate and attachment of filamentsoccurs directly on the free amino group of meso-diaminopico-late in a step catalyzed by the CapD transpeptidase (5). Here, we

show that CP5 of S. aureus Newman is released by mild acidtreatment from the cell wall. Furthermore, incubation ofmurein sacculi with endopeptidase or amidase, but not withglucosaminidase, released the capsular polysaccharide. Thesedata suggest that the staphylococcal capsule is attached to theglycan strands of peptidoglycan.

LCP proteins have been proposed to catalyze the transfer ofundecaprenyl phosphate-linked secondary cell wall polymerintermediates onto the amino sugar moieties of peptidoglycan(59). The typical structure of LCP enzymes is comprised of ashort N-terminal cytoplasmic tail, followed by 1 to 5 transmem-brane helices, and a C-terminal conserved domain (150 resi-dues) exposed on the bacterial surface and shared among allmembers of the LCP family (32). Based on crystallization stud-ies of S. pneumoniae Cps2A and B. subtilis TagT, the LCPdomain contains conserved residues important for coordinat-ing interactions with polyprenyl lipids, as well as Mg2 andpyrophosphate, and is endowed with pyrophosphatase activityin vitro (33, 59).

LCP-encoding genes often lie in close proximity to gene clus-ters encoding the biosynthesis of secondary polymers for whichthey exert their catalytic effects. For instance, in B. subtilis, thelcp genes, tagTUV, all fall within a 50-kb region on the chromo-some, directly adjacent to biosynthetic genes of teichuronicacid and minor teichoic acid, and within six open readingframes of the WTA gene cluster (59). Deletion of tagTUV is nottolerated and genetic depletion leads to loss of WTA (59). It isnot known whether LCPs attach teichuronic acids and minorteichoic acids to the envelope of B. subtilis. S. pneumoniaeencodes three lcp genes: cps2A, lytR, and psr, where cps2A is thefirst gene of the CPS operon. Disruption of cps2A alone or incombination with lytR results in reduced capsule synthesis, andrelease of CPS into the culture supernatants (33). S. agalactiaealso encodes three LCP genes, and cpsA is the first gene of thetype III capsule gene cluster. Deletion of cpsA results in reducedcell wall capsule (31). The contribution of LCPs to the group Bcarbohydrate of S. agalactiae has not been examined; assemblyof this carbohydrate, however, requires the enzyme GbcO, theTagO orthologue (58).

FIGURE 5. CP5 attachment to murein sacculi requires the LCP gene prod-ucts. S. aureus Newman wild-type (WT), tagO (WTA-deficient), capO (capsule-deficient), and LCP single (lcpA, lcpB, and lcpC), double (lcpAB, lcpAC, andlcpBC), and triple (�lcp) mutant without or with complementing plasmidsplcpA, plcpB, or plcpC were grown on Columbia agar supplemented with 2%NaCl. A, cells were harvested in 50 mM Tris-HCl (pH 7.5) buffer, normalized tothe same optical density, and suspension was fractionated into supernatant(S) and cells from which murein sacculi digested with lysostaphin (MS) werefurther extracted. The presence of CP5 in the supernatant and MS fractionswas examined by spotting samples on nitrocellulose and immunoblottingwith the �-CP5 antiserum. B, cell suspensions harvested and normalized as inA were fractionated into supernatant (S) or incubated with lysostaphin (T,total). Supernatant and total fractions were separated by SDS-PAGE andtransferred to PVDF membrane for Western blot analysis using the �-L6 anti-serum to assess for cell lysis during growth and document that roughly thesame quantity of cellular material were used for each strain examined. Num-bers to the left of the blots indicate molecular mass markers (kDa). C and D, CP5immunoreactive species in panel A were quantified by densitometry from atleast three independent experiments. Amounts of CP5 in WT samples werearbitrarily set as 1 and all other samples were normalized against wild typeCP5 levels. The white line marks the background level of CP5 immune reactivesignals. Statistical differences relative to WT (denoted by asterisks directlyabove the bars) or lcpC were determined by analysis of variance with a Dun-nett’s multiple comparison test (*, p � 0.05; **, p � 0.01; ***, p � 0.001).

FIGURE 6. LcpC is not preferentially produced when cells are grown incapsule-inducing medium. S. aureus Newman wild-type and �lcp strainswere cultured under standard laboratory growth conditions in tryptic soybroth (TSB) to mid-log phase (A600 � 0.5), or capsule-inducing conditions onColumbia agar supplemented with 2% NaCl (C/NaCl). Cells were harvested,normalized to A600 � 2, and lysed with lysostaphin. Proteins in these lysateswere separated by SDS-PAGE, transferred on PVDF membrane for Westernblot analysis using �-LcpA, �-LcpB, �-LcpC, and �-Sortase A (SrtA) antisera.Levels of SrtA were monitored to document that roughly the same number ofcells was used among strains examined. Numbers to the left of the blots indi-cate molecular mass markers (kDa). The data depict a representative of twoindependent experiments.




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S. aureus encodes three LCP genes, lcpABC. These genes arenot part of any biosynthetic cluster but deletion of any oneaffects attachment of WTA to the bacterial peptidoglycan (37).Staphylococcal WTA is a polymer of 30 –50 ribitol-phosphate(Rbo-P) subunits connected via 1,5-phosphodiester bonds (60).Rbo-Pn is tethered to peptidoglycan via the murein linkage unit,GlcNAc-ManNAc-(Gro-P)2–3 (61), sequentially synthesized byTagO (GlcNAc), TagA (ManNAc), and TagBDF (Gro-P) ontoundecaprenyl-phosphate (51, 62). The product of this pathwayis presumably flipped across the plasma membrane by theTagGH transporter (63). Attachment of the WTA polymer tothe C6-hydroxyl of N-acetylmuramic acid within peptidogly-can occurs during cell wall assembly (64) and requires at leastone LCP enzyme, as mutants with deletion of all three lcp genesno longer retain WTA in the envelope (37, 65). The lcpA andlcpB mutants display the largest reduction in WTA anchoring,whereas deletion of lcpC causes only a small reduction (37).Here, we show that lcpC encodes the key LCP enzyme forattachment of CP5 to the peptidoglycan of S. aureus. Althoughthe defect of the lcpC mutant is not further exacerbated inlcpAC or lcpBC double mutants, capsule attachment is entirelyabolished in the triple mutant (�lcp).

Wzy-dependent synthesis has been best described for assem-bly of O-polysaccharides in Gram-negative bacteria (11–13).This polymer is directly attached to lipid A and does not bear alinkage unit. The function of S. aureus capsular genes fits asimilar synthesis pathway with the exception of the last stepthat requires LcpC (Fig. 7). Considering the involvement ofLCP proteins in transferring polymers for which synthesis isinitiated by TagO (31, 37, 50, 51, 58, 59, 66, 67), we examinedwhether capsule assembly requires tagO in S. aureus. Further-more, recent work has shown that heterologous expression

of Pseudomonas aeruginosa O11 lipopolysaccharide synthe-sis genes, which include wbpL, a UDP-FucNAc-1-phosphatetransferase, together with CP5 genes capHIJK was sufficient tosynthesize lipid-linked CP5 polymer repeats in E. coli (68).WbpL is a protein that harbors the WecA/WbcO/MraY glyco-syl transferase homology domain, a TagO orthologue. Otherthan mraY and tagO, the S. aureus genome does not encode anyother genes bearing this conserved domain that could fulfill thisenzymatic role. Nevertheless, our data show that tagO is dis-pensable for CP5 synthesis, and thus we can exclude mureinlinkage units from the capsule assembly pathway. Interestingly,S. pneumoniae species do not encode tagO orthologues, otherthan mraY. The sugar transferase that initiates capsule synthe-sis by transferring glucose 1-phosphate onto the polyprenylphosphate lipid carrier is believed to be encoded by cspE/wchA,the fifth gene in the 17-gene cps/cap cluster (1, 69). Whenexpressed in E. coli, this gene is sufficient to synthesize a glu-cose-P-lipid intermediate (70). Deletion of this gene in S. pneu-moniae strains resulted in failure to elaborate a capsule (71). S.aureus capM encodes a protein with homology to WchA, andmay serve as the membrane-bound sugar transferase for thesynthesis of the UDP-FucNAc linkage (Fig. 7) (19). Althoughthis biosynthetic pathway seems plausible to us, experimentalproof for this model is not yet available.

Increased amounts of CP5 were observed when synthesis ofWTA was inhibited or abrogated. This result suggests that bothwall polymers may occupy the same C6 hydroxyl of N-acetyl-muramic acid or adjacent sites on peptidoglycan such thatoccupancy by one polymer precludes attachment of another.This phenomenon has been described for S. pneumoniae (1),where the absence/reduction of one polymer (WTA or CPS)leads to increased abundance of the other (72).

FIGURE 7. Model for S. aureus capsular polysaccharide synthesis. Enzymes unique to CP5 or CP8 synthesis are denoted. Enzyme names in italic refer to thosewhose roles are predicted based on homology and bioinformatics, and whose functions have not been experimentally validated. CapK is hypothesized to serveas a flippase, transporting the lipid-linked capsule intermediate from the cytoplasm to the extracellular milieu, whereas the function of CapC (not depicted) isunknown.




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In summary, we report here that CP5 is linked to the glycanstrands of S. aureus peptidoglycan, presumably to the same(C6-hydroxyl of MurNAc) or proximal sites as used for theanchoring of WTA. Capsule attachment is catalyzed by LcpC,albeit that LcpA and LcpB can functionally substitute at least inpart. Unlike WTA, capsule anchoring does not require TagO-mediated synthesis of murein linkage units (GlcNAc-ManNac)and likely utilizes undecaprenyl-PP-FucNAc, thought to beformed by a reaction that is catalyzed by CapM.

Acknowledgments—We thank Yimei Chen for assistance in transmis-sion electron microscopy, and Andrea DeDent, Matthew Frankel, andVilasack Thammavongsa for critical discussion.

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Yvonne Gar-Yun Chan, Hwan Keun Kim, Olaf Schneewind and Dominique MissiakasPeptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Is Attached toStaphylococcus aureusThe Capsular Polysaccharide of

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