The key role of peptidoglycan in theopsonization of Staphylococcus aureus.

P K Peterson, … , P G Quie, J Verhoef

J Clin Invest. 1978;61(3):597-609. https://doi.org/10.1172/JCI108971.

In an effort to determine the staphylococcal cell surface component(s) of importance inopsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolatedfrom Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubationof the cell walls and peptidoglycan with various opsonic sources, uptake by humanpolymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosisof cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removalof teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of theparent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and boththe classical and alternative pathways participated in opsonization. Kinetic studies revealedthat opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan,proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when theclassical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed withpeptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involvedin activation of both the classical and alternative complement pathways. Thus,peptidoglycan appears to be the key cell wall component involved in staphylococcalopsonization, and it is suggested that host […]

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The Key Role of Peptidoglycan in the Opsonization of

Staphylococcus Aureus

PHILLIP K. PETERSON, BRIAN J. WILKINSON, YOUNGKIKIM, DAVID SCHMELING,STENVEND. DOUGLAS,and PAULG. QUIE, the Departments of Medicine, Pediatrics,atnd Microbiology, University of Minnesota School of Medicine, Minneapolis,Minnesota 55455

JANT VERHOEF, Laboratory for Microbiology, Utrechlt, Netherlands

A B S T RA C T In an effort to determine the staphylo-coccal cell surface component(s) of importance inopsoniization, cell walls (peptidoglycan and teichoicacid) and peptidoglycan were isolated from Staphylo-coccus aureus strain H grown in [3H]glycine-con-taining broth. After incubation of the cell wallsand peptidoglycan with various opsonic sources,uptake by human pol'ymorphonuclear leukocyteswvas measured. The opsonic requirements for phago-cvtosis of cell walls and peptidoglycan were foundto be similar to those of intact bacteria. Removalof teichoic acid from the cell wall did not affectopsonization. Likewise, a teichoic acid-deficient mut-ant strain of S. aureus H was opsonized in a mannersimilar to that of the parent strain. Immunoglobulin Gfunctionied as the major heat-stable opsonic factor andboth the classical and alternative pathways participatedin opsonization. Kinetic studies revealed that opsoniza-tion of peptidoglycan, as well as C3-C9 consumptionby peptidoglycan, proceeded at a slower rate via thealternative pathway (C2-deficient serum) than whenthe classical pathway was present (normal serum). Theability of peptidoglycan to activate C3-C9 was sig-nificantly reduced when normal and C2-deficient serawere preabsorbed with peptidoglycan at 2°C suggest-ing that antibodies to peptidoglycan may be involved inactivation of'both the classical aind alternative comple-menit pathways. Thus, peptidoglvcan appears to be thekev cell wall comiipoineint iinvolved in staphylococcalopsonlizationi, aind it is suggested that host response topeptidlogl-can, a major cell wall component of mostgramn-positive bacteria, max be related to the develop-miienit of i'natniral imm-niunity" to this group of mnicroor-ganlism11s.

Dr. Qoiie is the Aier-icani Legion Memorial Heart ResearchProfessor.

Rece ied for publication 4 August 1977 aoid in revi.sedfor1;127 October 1977.

INTRODUCTION

In recent years increased attention has focused on thebiological significance of the cell walls of gram-positivebacteria. In particular, peptidoglycan, a major compo-nent of the cell wall of most gram-positive bacteria, hasbeen shown to play a significant role in determining thenature of host response to this group of bacteria (1, 2). Inthe case of Staphylococcus aureus, this cell wallcomponent has been demonstrated to possessendotoxin-like properties (3), to activate the serumcomplement (C) system (4), and to elicit a cell-mediatedimmune response (5, 6). Few studies, however, haveexamined the role of the cell wall components of S.aureus in the process of opsonization, and results havebeen conflicting regarding the relative importance ofthese components in this process (7-12).

In this investigation the role of the two majorcomponents of the S. aureus cell wall-the peptidogly-can and teichoic acid-in the process of opsonizationwas studied by measuring the uptake of radioactivelylabeled bacteria and purified cell wall components byhuman polymorphonuclear (PMIN)' leukocytes. Thenature of the opsonic factors involved in this processand the kinetics of C activation were examined. Data ispresented which demonstrates that opsonization byboth heat-stable (IgG) and heat-labile serum factors ismediated by cell wall peptidoglycan. Peptidoglycanwas found capable of activating both the classical andalternative pathways of the C system. It is proposed thatCl may' play a role in activation of the alternativecomiiplenmeint pathway by peptidoglycan and that activa-tion of both the classical aind alternative pathways isinitiated by antigen-antibody comiiplex formation.

Abbreviatiotis utsed in this pa per: CH5(,, total liemol ticcompleim1ent; HBSS, Han1ks' balaiced salt solutioin with 1%gelatini; PBS, phosphate-buffered saline; P.MN, polvmor-phon icleair.

The Jouirtnal of Clintical Itncestigation Volutmne 61 Marchi 1978 597-60(9 597

METHODS

Bacterial strainsS. aureus strain H, choseen because of its well-estal)lished

cell wall structure (13), was kindly provided by Sir JamesBaddiley's laboratory, University of Newcastle-upon-Tyne,England. S. aureus HSImIR (a spontaneous streptomycin-resistant isolate of strain H) and a teichoic acid-deficienitmutant, straini 52A5 (14), were generously provided by J. T.Park, Tufts University, Boston, Mass. The cell wall of strain52A5 appears to be composed almost entirely of peptidoglycan(15). The organisnms were maintained on Trypticase Soy Agar(Difco Laboratories, Detroit, Mich.) slants.

Cultivatiotn and radioactive labeling ofbacteriaBacteria were grown in peptone yeast extract broth (16) with

aeration on an orbital shaker at 37°C. For phagocytosis studiesof intact bacteria, the organisms were labeled by inocuilatinigseveral coloniies of bacteria into 20 nml peptone yeast extractbroth containing 40 ,uCi [2-3H]glycine (sp act 5-15 Ci/mmol,NewEngland Nuclear, Boston, Mass.). After an 18-h incubationat 37°C, the bacteria were washed three times in phosphate-b6uffered saline (PBS), pH 7.4, and resuspended in PBS to afinal concentration of 1 x 109 colony-forming units/ml using aspectrophotometric method confirmed by pour plate colonycounts. For preparing radiolabeled cell walls, organisms weregrown in peptone yeast extract broth, 2 liters containing[2-3H]glycine, 0.4 ,uCi/ml, and were harvested in lateexponential growth phase (OD625 nm 1.5-2.0, Spectronic20, Bausch & Lomb Scientific Optical Products Div., Roches-ter, N.Y.). Large scale growth of unlabeled bacteria (26 liters)was achieved in a NewBrunswick Fermentor (New BrunswickScientific Co., Inc., NewBrunswick, N.J.) at 370C with forcedaeration (5 liters/min) usinlg anitifoam (Anti-Foamii aerosolspray, Sigimia Chemical Co., St. Louis, Mo.). 2 liters of a 15-hculture was used as an inoculum, and the bacteria were grownfor 6-7 h and harvested at an OD625 nm of 1.0-1.5. Thecultures were harvested in a continuous flow centrifuge(Beckman J-21) using a JCF-2 rotor (Beckman InstrumentsInc., Spinco Div., Palo Alto, Calif:) operating at 2,500 g with aflow rate of 250 ml/min. The organisms were washed once incold distilled water and then frozen (- 15°C) overnight beforedisruption. The yield was 80 g wet weight of cells from 26liters of culture.

Isolation of cell walls, peptidoglycan, andteichoic acidUnless otherwise indicated, all operations were carried out

at 40C and all centrifugations were at 16,319 g for 10 min at 40C.Washed organisms in distilled water were mixed with glassbeads (0.10-0.11 mm, VWRScientific Div., UNIVAR, SanFrancisco, Calif.), and disrupted by agitation in a VibrogenCell Mill (R.H.O. Scientific Inc., Commack, N. Y.) for 20 min atmaximum speed. The glass beads were removed by filtrationon a coarse glass sinter and the filtrate was centrifuged todeposit the white cell walls overlaying yellower, unbrokenorganisms. Care was taken to avoid resuspending unbrokenorganisms. The crude cell walls were washed once in distilledwater, resuspended in 200 ml 2% sodium dodecyl sulphate,and then stirred overnight at room temperature to inactivateautolytic enzymes and to aid in stripping of membrane

comlponents. The cell walls were then washed four times inwater at roomii temperature before being resuspended in 200 ml0.05 M Tris-HCI containing MgCI2 (5 mM) plus 2.5 ,ug/mleach of RNase (EC 3.1.3.4, 2,588 U/mg) and DNase (EC3.1.4.22,2,500 U/mg) (each type 1 from Worthington Biochem-ical Corp., Freehold, N.J.) and incubated at 37°C for 1 h withgentle shakinig (100 rpm). To remove proteinaceous materialincluding protein A (17), 200 ,ug/ml, trypsin was added (EC3.4.4.4, 189 U/ml, Worthington Biochemical Corp.), and theincubationi contintued for 4 h. The walls were washed once inwater and then stirred with an equal volume of 80%phenol for30 lnin at room temperatuire to remove lipoteichoic acid (18).After extraction, the walls were washed six times in water andthen lyophilized. 1,680 mgof cell wall was recovered from 26liters of culture.

Cell walls (1,260 mg) suspended in 75 ml water were mixedwith 75 ml of 20% (wt/vol) TCA and were incubated at 60°Cfor 90 min to remove teichoic acid (19). The suspenision wascenitrifuged to yield peptidoglyean which was washed fourtimes with water before being lyophilized (yield 1,067 mng,35.5% weight of wall removed.) Teichoic acid was recoveredfrom the suiperniate by adding 750 ml cold EtOH and allowingto stand at 4°C overnight. The precipitate recovered bycentrifugation was resuspenided in 14 ml 10% TCA andallowed to precipitate again overnight at 4°C with 70 ml EtOH.The product was washed twice with acetone and allowed to airdry.

Typically, cell walls were labeled to a specific activity of-5 x 105 cpiml/milg dry weight. The hot TCAtreatment resultedin about 10% loss of counts indicating little solubilization ofglycine bridges occurred (20) and the specific activity wasroughly dcouble that of the cell walls. Preparations were storedfrozen at - 15°C at a concentration of 5-10 mgdry weight/nil,and brief treatment in a soInic bath (Branson Sonic Power Co.,Danbury, Conn.) was used to help attain an even suspensionbefore each opsonization procedure.

Chemical characterization of cell walls,peptidoglycan, and teichoic acid

Quantitative examination. Carefully weighed cell walls,peptidoglycan, and teichoic acid were hydrolyzed in sealedtubes with 6 MHCI for 18 h at 105°C. Acid was removed undervacuum over anhydrous CaCl2 in the presence of NaOHpellets; the dried residues were twice moistened with 0.5 mlwater and redried. Amino acids and amino sugars wereestimated on an aminio acid analyzer (Beckman model 120BAmino Acid Analyzer, Beckman Instruments, Fullerton, Calif.Inc.,). Phosphoruis was estimated by the method of Fiske andSubba Rowas described by Leloir and Gardini (21). Total fattyacids in 10-mg amounts of preparation were determined asdescribed by Wilkinson (22).

Qualitative examination. Teichoic acid (5 mg) washydrolyzed in 2 MHCI for 3 h at 105°C in sealed tubes; acidwas removed as described above. Part of the hydrolysate wastreated with alkaline phosphatase (EC 3.1.3.1, 1 mg, type 1,calf intestinal mucosa, Sigma Chemical Co.) in 0.025 MNH4HCO3 containing 0.005 M NH40H, pH 8.3, over-night at 37°C. Alkaline phosphatase-treated and untreatedhydrolysates were examined by descending chromatog-raphy on Whatman Ino. 1 paper in the solvent systems 6propan-1-01:3 0.88 NH3: 1 H,O (by volume) and 6 butan-1-01:4pyridine: 3 H20 (by volume) (23). Reducing compounds weredetected with the alkaline AgNO3reagent (24) and compoundswith amino groups by ninhydin treatment (0.25% wt/vol inacetone plus heating at 100°C for 5 min).

598 Peterson, Wilkinson, Kim, Verhoef, Schmeling, Douglas, and Quie

Opsonic soU rces

Serum was pooled from five healthy donors who cleniiedprevious staphylococcal infection (normnal serum). To blockC I, normal serumn was chelated with ethylene gly cotetraaceticacid in the presence of MgCI2 (MgEGTA) in a 10-M to 10-mMconcentration with respect to serum (25). C2-deficient seruml]was obtained froml a patient with genetically determinedcomplete albsence of C2 and normal levels of all other Ccomlponents. Serum was stored in 0.5-mll ali(luots at -70°C.Heat-iniactivated serum was prepared by heating thawedaliqtuots of serumil at 56°C for 30 mni. Purified humani IgG wasprovicled bh Dr. D. Roos, Central Laboratory of the BloodTransfusion Service, Amsterdanm, Netherlanids. Serum andIgG were diluted to specified coneentrations with Hank'sbalanced salt solution conitaining 1% gelatin (HBSS) justbefore use. Calciumil- andl magnesium-free HBSSwas usedl to(lilute M\gEGTA-chelated serum.

Opsonization procedureAccording to Chatterjee (26), _1 x 108 whole organismiis

yield 25 Ag of cell walls. Conise(luently, 0.1-mI ali(quots ofintact bacteria (1 x 1)8 colony-forming units) and 25 lig of cellwalls were used in all opsonizationi experimiienits. Studies tisiig2.5 and 12.5 jg of peptidoglycan yielded similar find(lilgs;results were reported using 25 Ag of peptidoglycan. Eachaliquot was incubated at 37°C in a 12 x 75-mm plastic tube,BioQtuest, BBL & Falcon Products, Becton, Dickinson & Co.,Cockey sville, Md., containing 1.0 ml of the indicated opsonlicsource for specified tinmes followed by centrifuiginig at 4,000 gfor 15 min (4°C). The supernate was discarded and the pelletresuspeinded in 0.5 ml HBSS.

PMNleukocytesBlood from healthy donors was drawni into heparinlized

syringes (10 U heparin/ml blood). PMIN leukocytes wereprepared using a method modified from B16yum (27) in whichthe blood was centrifuged for 15 mmi at 200 g, and theplasimia-platelet layer was removecd with a Pasteur pipette. Theleukocvte layer was then withdrawn, placed in plastic tubes,and then diluted with two parts Spinner's Minimal EssenitialMedium (iX, Grand Island Biological Co., Grand Island, N. Y.).8 ml of this suispenisioni was carefully layered onto 3 mlFicoll-Isopaque (lymphocyte separation medium, LittonBionetics, Kensinigton, Md.). After centrifuging at 200 g for 30mmin the mononuclear cell laver was removed with a Pasteurpipette. To obtain pure PMN leukocytes, the remainingmedium was withdrawni and discarded, and 0.87% NH4Cl insterile water was added to the tube containing theerythrocyte-PMN leukocyte pellet which was then rotated for10 min at room temperature. After centrifuginig for 10 min at160 g, the PMN leukocyte pellet was washed an additional twotimes with 0.87% NH4CI to lyse the renmaining erythrocytes.The PMNleukocytes were resuspended finally in HBSSandtotal and differential leukocyte counts were performed. Thefinal leukocyte pellets were adjusted to a concentration of 1(7PMNleukocytes/ml HBSS. Contamination by mononuclearcells was evaluiated by Wright's stainied smears and generallydid niot exceed 1%.

Phagocytosis mixtures and assaysTo each 0.5 ml suspension of opsonized radioactivelv

labeled intact bacteria, cell walls, and peptidoglycan, 0.5 ml of

PMNleukocyte suspension was added. These mixttures wereincubated at 37°C in an incubator shaker (New BrunlswickScientific Co., Inc.) at 250 rpm. PMN leukocyte uptake ofbacteria, cell walls, and peptidoglycan was quantitated using amodificationi of a previously described method (28). Briefly,leukocyte-associated radioactivity was determined by takingduplicate 100-,ul samples from each phagocytosis mixture atspecified time intervals, placing the samples in 3 ml PBS, pH7.4, in polypropylene vials (Bio-vials, Beckmiiani InstrumnenitsInc.,) and washing the leukocytes three times in PBS bydifferential centrifugation at 160 g. Final pellets were resus-pended in 3 ml scintillation li(quid (toluenie containinlgfluorallov [TLA, Beckman Instruments, Inc.] and 20%Biosolv-3 [Beckman Instrunmenits, Inc.]) and couniited in aliquid scintillation counter (Beckmain LS-250). To determinetotal radioactivity (representing both leukocyte-associated aoldnonleukocvte-associated bacteria, cell walls, or peptidoglv-cai), duplicate lO0-pl samples were placed in 3 ml PBS at theend of the assay period and centrifuged at 4,000 g for 15 min.Supernates were discarded, pellets resuspended in scintilla-tion liquid, and samples counted in the scintillation counter. Anaverage of duplicate values was used in all calculationis. PMNleukocyte uptake of radioactively labeled bacteria, cell walls,and peptidoglveacni at given sanmpling times was calculatedusing the formula: %Nuptake = (cpm in leukocyte pellet)/(totalcpmli) x 100.

To study opsoniization of soluble peptidoglycan fragnmenits,purified peptidoglycan was digested with the muralvticenzyme, lysostaphin. 0.5 mg (0.1 ml) peptidoglycan wvasinicubatedl in 0.9 ml PBS containing 10 gg/ml lvsostaphini(Schwartz Mann Div., Becton, Dickinson & Co., Orangeburg,N.Y.) for 60 min at 370C followed by centrifuging at 10,000 g for30 min. Suipernates were then heated in a boiling water bath for15 min to inactivate lysostaphin. 50-,ul aliqIuots of the stipernate(peptidoglycan fragments) were then incubated with opsonlicsources for 15 min. PMNleukocytes were added, and uptakewvas determined as described above. Total radioactivity inthese mixtures was determined by suspendinig 100-,ul salmplesdirectly into sciintillation liqiuid. A control mixtuire of 0.5 mgpeptidoglycan was incubated in PBS for 60 min; aftercentriftiging, the supernate was discarded and the pelletresuspenided in 1.0 ml PBS containing 10 ug/ml lysostaphiniwhich had been previously heat-inactivated. This mixture wasthen heated in a boiling water bath after which 50-,l ali(quotswere removed for opsonization and phagocytosis studies.

Measuremnent of C activation by purifiedpeptidoglycan0.1-ml ali(qtuots of peptidoglycani, at specified conicenitra-

tions, and of water (control) were added to 12 x 75-mmplastic tubes (BioQuest, BBL & Falcon Products) conl-taining 1.0( ml normal serum. After a 30-mmi incubation at37°C, the tubes were centrifuged at 4,000 g for 15 min (4°C) andsupernates were stored at -700C before measurement of totalhemolytic complement (CH50).

To study the kinetics of C activation in normal serum and inC2-deficient serum, 0.1-ml (1.0 mg) aliquots of peptidoglvcanwere added to 1.0-ml samples of serumn followed by incubatingat 37°C for indicated time intervals. Control mixturescontaining 0.1 ml distilled water were incubated simultane-ously and removed at the same time intervals. After cenitrifug-ing at 4,000 g for 15 min (4°C), the supernates were stored at-70°C before measurement of C3-C9.

The effect of preabsorption of normal and C2-deficient serawith peptidoglyean on the subsequent ability of peptidoglycan

Opsonization of Staphylococcal Peptidoglycan 599

to activate the serum C system was studied by incubating1.0-ml sami-ples of serum three times for 60 min at 2°C witheither 0.1-ml (1.(0 mg) aliquots of peptidoglycan or 0.1-milaliquots of distilled water (nonabsorbed serum). After each60-min absorptioni period, samples were centrifuged for 15min at 4,000 g (2°C) and the supernatant serum was added tothe next precooled tube conitaininig either peptidoglycan orwater. After the final centrifugation, the serum samples wereadded to tubes containing 0.1-ml aliquots of peptidoglycan, atindicated concentrations, and were then incubated at 37°C forspecified time intervals. Control tubes containing absorbedand nonabsorbed serum and 0.1 Iml distilledl water werelikewise incubated at 37°C. After incubation, samples werecentrifuged at 4,000 g for 15 min (2°C) and the supernates storedat -70°C before C3-C9 determination.

Hemolytic titrations of C were performed using sheeperythrocytes in acid citrate dextrose (Wilfer Laboratories,Stillwater, Minn.). Intermediate cell preparation (C14) and theassays of CH50 and C3-C9 were performed as previouslydescribed (29-32). For CH50, the optical density was read at541 nm and for C3-C9 titration at 412 nm. Results wereexpressed as 50% lysis, and titrations were corrected forday-to-day variations by including a knowin internal standardhuman serum.

Electron microscopylJCell walls and peptidoglycan were examined by a Siemens

102 electron microscope (Siemens Corp., Medical/IndustrialGroups, Istlin, N. J.) using previously described methods (33).In addition, 0.1-ml aliquots of peptidoglycan (1 mg) wereincubated in plastic tubes (BioQuest, BBL & Falcon Products)containing 1.0 ml 10% normal serum, 10% heat-inactivatedserum, and HBSS for 60 min. After centrifuging for 15 min at4,000 g, supernates were discarded and 0.5 ml HBSSwas addedto each tube. Leukocyte suspensions (0.5 ml) were then addedand the mixtures incubated for 15 min at 37°C. The leukocyteswere washed three times with cold PBS, fixed in 1.5%glutaraldehyde in PBS, and then thini sections were examined.

RESULTS

Chemistry of the cell walls, peptidoglycan, andteichoic acidTable I shows the quantitative analysis for the cell

wall preparations used in this study. It can be seen thatamino acids other than those normally considered to bepart of the staphylococcal peptidoglycan were eithernot detected or present in only small amouints. The lackof the full range of protein amino acids indicates littlecontamination of the cell walls with proteinaceousmaterials including protein A. On the basis of theanalyses presented here, and knowledge of the S.aureus peptidoglyean structure (19, 34), it can be seenthat the average subunit of the peptidoglycain containisisoglutanmine, 1.0; lysine, 1.0; alanine, 2.0; glycine, 4.0;serine, 0.1; N-acetylmuramic acid, 1.0; N-acetylglucos-amine, 1.0; O-cacetyl, 0.6; aind the formula Nweightis 1,138. Using the lysine value it cani be seenthat 50.5% of the dry weight of the wall isaccouinted for by the peptidoglycan. For isolatedpeptidoglycan, 92.4% of the dry weight of thepreparation is accountal)le for as peptidoglycan com-

TABLE IChenmistry of Cell Waoll.s, Peptidoglicat, (al)1 'Tichoic Acid8

Conlipolnelnt Cell walls Peptidoglycand Teicloic acid

Muramic acid 180.4 280 TraeGlucosanmine 682 412 1,560Glutanmie acid 277 668 24Lysine 444 812 48Alanine 849 1,588 :310Glycine 1,826 3,460 120Seriine 48 68 -

Phosphate 1,((( 84 2,000Aspartic acid 26.4 24Threonine 8.8 8 --Valine 8.8 8Isoleucine 8.8 8Leucine 8.8 8Histidiine 30.8 20Arginine 8.8 16

* Cell walls, peptidoglINcain, and teichoic acid wvere isolatedlfrom S. aureus strain H.

ponents. Assumiiing that ribitol phosphate and N-acetyl-glucosamine are equimolar then one can take ribitol,1.0; phosphate, 1.0; N-acetylglucosamine, 1.0; alanine,0.16 as the average teichoic acid sub-Un1it (see lelow)and this gives a formula weight of 410. Thus, 41%of thedry weight of the cell wall is present as teichoic acidplus 50.5% as peptiodglycan thereby accounting for91.5% of the dry weight of the cell wall. It is likely thatmetal counterions to negatively/ charged groups in theteichoic acid and peptidoglycan mlake up aconsiderable portion of the uinaccounited wveight ofthese preparations (35). 82% of the teichoic acidpreparation is accountable for as teichoic acidlcomponients.

Additional evidence that the peptidoglycan is free ofteichoic acid is the finding that 95% of the cell wallphosphorus was renmoved by the hot TCAextraction andlthe ratio of the glucosamine to muramic acid chanigedfrom an excess to being almost e(uimiiolar.

Qualitative examination of the teichoic acid hydroly-sate revealed components with chromatographic m11o-bilities characteristic of glucosamiiiie, ribitol, altnine,glucosaiminitol phosphaite, ribitol di- aiud inoniophos-phate, and glucosaminitol ribitol. Amino acid analysesof the teichoic acid revealed glticosamine anld alanineto be the imnajor componients. The al)sence of miiramnicacid andl the low amounts of other a-n)ino iacids showthat the teichoic acid fratctioni does nlot conitaii nmajoramounts of peptidoglyca.n In .an effort to (leteit- rincw,hether the cell wvall aind peptidoglycan pre piar.tionswere contami-niated with lipoteichoic acid, the totalf:atty ateid comiipositioni of the prepiarations \wTajSdleternii ined. Fatty atci(ls ar-e imarkers of eel1 ineihl)ran e

600 Peterson, Wilkin son, Kitl, Verhoef, Sclhlneling, Douglas, andlI (Jo ie

a

C s a i MRFIGURE 1 Electroni micrographs of cell w alls, (a ) x20,265, (b) x 100,000 and peptidogl can, (c)x20,2565, (d) x 100,000 isolate(d fromii S. atireutsl H.

lipids and/or lipoteichoic acid. This was comparedl withthe fattv acid compositioni of"crude cell walls" that hadnot been treated with sodiumil dodecvl sulfate, phenol,

or enzvnmes but had simply been washed six times inwater. Five major fatty acid methvl ester peaks wereobtained from the crude cell walls by gas-liquid

Opson ization of Staphylococcal Peptidoglycan 6601

4mohn

ir

chromatography. Chromatography of the fatty acidmethyl ester fraction from an equal weight of thepurified cell walls used in this study revealed only onevery small "peak" on the trace that was difficult toestinmate. Similar examination of the peptidoglycanrevealed two extremely small peaks. Summation of thetotal detector response revealed that the cell walls andpeptidoglycan had at least 97.4 and 98.7%, respectivelyless fatty acids than the crude cell walls. The cell wallanid peptidoglycan preparationis were contaminatedwith fattv acids at levels < 1 nmol/mg. These results,plus the fact that we cani account for most of the weightof the preparations in known conmponents as well as theproveni value of sodium dodecyl sulfate and phenolextractions in renioving lipoteichoic acid (18, 36),indicate the preparations are not significantly con-taminated with lipoteichoic acid.

Thus, the qualitative and (uantitative analysesindicate that the cell walls (composed of peptidoglycanand teichoic acid) and peptidoglycan used in thisinvestigation are highly purified and chemically consis-tent with the observations of other investigators (15, 34).

Electron micrographs of glutaraldehyde-osmiumtetroxide-fixed, embedded sections of cell walls andpeptidoglycan are shown in Fig. 1. The preparations are(luite homogenous and devoid of whole organisms anddebris. Both cell walls and peptidoglycan reveal acharacteristic trilaminar appearance and septa (37). Themorphologic appearance of the peptidoglycan is some-what collapsed and folded in comparisoni to the cellwalls. In general, the peptidoglycan is thinner insectionied material than the cell walls, a finding inagreemeint with previous studies (37, 38); detailedmorphometric measuirements, however, were not per-formed.

Phlagocytosis of S. aureus H and of isolated cellwalls and peptidoglycanKinetics of uptake by PMN leukocytes. Intact S.

aureus H, isolated cell walls (peptidoglycan withcovalently linked teichoic acid) and purifiedpeptidoglycan were incubated with 1% normal serum,1%heat-iniactivated serum, and HBSSfor 15 min beforeadding PMNNleukocytes. As shown in Fig. 2, uptake ofintact bacteria, cell walls, and peptidoglycan wassimiiilar at 3, 10, and 15 min. At 15 min, -70% of each of'the three types of"particles" opsonized with normal serumwere letkocyte-assoc iated compared with _25% andvirtually nlo uptake of particles which had beenincubated with heat-inactivated sertum and HBSS,respectively. These restults stuggested that removal ofteichoic acid fronm the cell wall preparations did notsign ificantly alter opsonizationi aind phagocytosis byPMNleuikocytes.

To determine whether levikocyte-associated radioac-tivity represented attached and/or inigested peptidogly-

100_

80 _ .cwit.orNS

60

iOO 40 - S.ourPG AS

20-c

Sourcw HBSS

0 3 10 15Minutes

FIGURE 2 PMNleukocyte uptake of intact S. aureus H (S.aur), isolated cell walls (CW) and purified peptidoglyean (PG)preincubated with 1%normal serum (NS), 1%heat-inactivatedserum (AS) and Hanks' balanced salt solution (HBSS) beforeadding leukocytes and determining uptake at 3, 10, and 15min. Results represent means of three experiments.

can, electron microscope examination was performed(Fig. 3). After opsonization with normal serum, pep-tidoglycan was present within phagocytic vacuoles inthe leukocytes and significant degranulation has oc-curred. When preincubated with HBSS, essentially nopeptidoglycan was found within the leukocytes. Op-sonization with heat-inactivated serum resulted in lessingestion than when normal serum was used as anopsonic source but in greater ingestion than when noopsonin was present (HBSS).

Opsonic titer of normal and heat-inactivated sera.When 1% serum was used as an opsonic source PMNleukocyte uptake of cell walls (peptidoglycan withteichoic acid) and peptidoglycan was found to besimilar. This result, as stated above, suggested thatremoval of teichoic acid from the cell wall did notsignifiantly affect opsonization. To test the possibilitythat removal of teichoic acid might change the titer ofserum capable of effective opsonization, intact bacteria,cell walls, and peptidoglycan were incubated for 15 minwith varying concentrations of normal serum andheat-inactivated serum before adding leukocytes anddetermining uptake at 15 min (Fig. 4). Phagocytosis ofall three particles was significantly reduced and to thesame extent when 0.1% normal serum and when 1.0%heat-inactivated serum were used as opsonic sourcesthereby demonstrating that the opsonic titer was notinflueniced by the removal of teichoic acid from the cellwall. When purified human IgG, in concentrations of2.5, 1.0, 0.1, and 0.01 mg/ml, was used as an opsonicsource, uptake was found to be similar to that of 5.0, 1.0,0.1, and 0.01% heat-iniactivated serum, respectively(data not shown). This suggested that IgG was the majoropsonic factor in heat-inactivated serum and implies

602 Petersoni, Wilkin.son, Kim, Verhoef, Schmeling, Douglas, a(nd Quie

eAir- IA. I

e- ".I

4h i", . A_::;---%.

1, 'Wrol:.,,-F't. 1-

FIGURE 3 Electron micrographs of peptidoglyean after a 15-min incubation with PMNleukocytes. Peptidoglycan preincubated with HBSS remains extracellular, (a) x7,500; peptido-glycan preincubated with normal serum is present within phagocytic vacuoles and degranulationhas occurred, (b) x7,500, (c) x50,OOO.

that purified human IgG contains antibodies against though the results of the above experiments suggestedpeptidoglycan. that peptidoglyean was the major component involved

Phagocytosis of S. aureus HSmRand 52A5. Al- in opsonization by normal and heat-inactivated sera,

Opsonization of Staphylococcal Peptidoglycan

0 :-

4. ib -0

.0-1.7 .PfN-

..4" 1m 11

-, .1.f!V1. 4

-1. .K,

603

I)80 * S.aoureus H0 Ce/f Wo/lsc~~~~~~~~~~ ~~~Pep/ldoglycan

E 60

4)40

0

501.00.10.01 50 1.O l 0.01 5D I1 0001 5D 1.0 0.10.01 5D 1.0 01 0.01 50 1.00.10.01

Serum Concentration (%)

FIGuRE 4 PMN leukocyte uptake at 15 min of intact S.aureus H, isolated cell walls and purified peptidoglycanopsonized with varying concentrations of normal serum (A)and heat-inactivated serum (B). Results represent means ofthree experiments.

and that cell wall teichoic acid played little or no role inthis regard, electron microscope studies of thin sectionsof cell walls and peptidoglycan (Fig. 1) had indicatedthat these preparations differed morphologically aswell as in their previously defined chemical composi-tions. Therefore, it seemed possible that removal ofteichoic acid from the cell wall might have affectedopsonization independently of the change broughtabout in chemical makeup. To investigate this possibil-ity, opsonization of a teichoic acid-deficient mutant(52A5) was compared to that of its parent strain (HSmR)(Fig. 5). As shown, phagocytosis of the teichoic

A. Normol Serum B. Heat-inactivated Serum100

Parent StrainInstan) Tn25(ecocai-elchcintlmutant)lpreincubate

80

U-) 60

(D

O' 20

5.01.0 01 5.01.0 01 5.01.0 01 5.0100.1

Serum Concentration (%FIGURE 5 PMNleukocyte uptake of S. aureus HSMR(parentstrain) and 52A5 (teichoic acid-deficient mutant) preincubatedwith varying concentrations of (A) normal serum and (B)heat-inactivated serum before adding leukocytes and deter-mining uptake at 15 min. Results represent means of threeexperiments.

acid-deficient mutant did not differ significantly fromthat of the parent strain when varying concentrations ofnormal and heat-inactivated sera were used as opsonicsources. This result provided further evidence that theteichoic acid component of the cell wall did not have amajor influence on bacterial opsonization. After eachexperiment the typeability of the strains was checkedwith phage 52A (39). Teichoic acid forms part of thereceptor site for this phage (14). The parent organismremained susceptible to this phage and strain 52A5 wasnot susceptible, thereby inidicating that reversion of the52A5 strain to a teichoic acid-containing form had notoccurred.

The role of Cl and C2 in opsonizationi. To study therole of the classical C pathway in opsonization, Cl wasblocked by chelating normal serum with MgEGTA,andopsonization in the absence of C2 was studied by usingserum from a patient with complete C2 deficiency.Intact bacteria, cell walls, and peptidoglycan wereincubated for 60 min with 1%concentrations of normal,heat-inactivated, MgEGTA-chelated, C2-deficient, andheat-inactivated C2-deficient sera before adding PMNleukocytes and measuring uptake at 15 min. Normalserum was the most effective opsonic source for intactbacteria, cell walls, and peptidoglycan, uptake beingsignificantly greater than when either MNgEGTA-

100r-

InU)-

cL

0-)0

Q.

*d S. aureus H

O3 Ce// Wa//sES Pepildog/ycan

80K

60K

40 H

20 H

0'def def def def

Opsonic Sources

FIGURE 6 PMN leukocyte uptake of intact S. aureus H,isolated cell walls, and purified peptidoglyean preincubatedfor 60 min with normal serum (NS), MgEGTA-chelated serum(X), heat-inactivated serum (AS), C2-deficient serum (C2 def)and heat-inactivated C2-deficient serum (AC2 def) beforeadding leukocytes and determining uptake at 15 min. Resultsrepresent means of five experiments. Significance of differ-ences in uptake comparing NS and X, P < 0.001 for intactbacteria, cell walls and peptidoglycan; comparing NS and C2def, P < 0.025, 0.001, 0.001 for intact bacteria, cell walls andpeptidoglycan respectively; comparing C2 def and AC2 def,P < 0.001 for intact bacteria, cell walls and peptidoglyean (ttest for paired data).

604 Peterson, Wilkinson, Kim, Verhoef, Schmeling, Douglas, and Quie

chelated or C2-deficient sera were used as opsonicsources (Fig. 6). Although MgEGTA-chelated serumwas not significantly more opsonic than heat-inactivated serum, C2-deficient serum had a greateropsonic capacity than heat-inactivated C2-deficientserum.

These results suggested that opsoniization couldproceed via a heat-labile pathway other than theclassical pathway when C2 was blocked but not whenCl was blocked. When the period of opsonization waslimited to 15 min rather than 60 min, however,C2-deficient serum was no more effective than heat-inactivated C2-deficient serum as an opsonic source,opsonization with the other serum sources not beingaffected by the shorter opsonization period (data notpresented). This finding indicated that opsonization viathe second heat-labile pathway proceeded more slowlythan when opsonization was carried out in the presenceof the classical pathway.

Effect of lysostaphin treatment of peptidoglycan onopsonizationi. To determine whether relatively small,soluble fragments of peptidoglycan could be opsonizedby normal and heat-inactivated sera, peptidoglycan wastreated with lysostaphin and the fragments incubatedwith 10% normal serum, 10% heat-inactivated serum,and HBSSbefore adding PMNleukocytes and measur-ing leukocyte-associated radioactivity at 3, 10, and 15min. Whereas there was uptake of intact peptidoglycanin the control mixtures similar to that observed inearlier experiments (Fig. 2), no leukocyte-associatedradioactivity was recorded when peptidoglycan frag-ments were studied. These results suggested that foreffective opsonization a certain particle size of thepeptidoglycan was required.

C activation by peptidoglycanEffect of peptidoglycan concentration on C activa-

tion itn niormal serum. As results from the aboveexperiments indicated that the peptidoglycan of S.aureus H was the major C-activating component of thecell wall, additional studies were performed in whichconsumption of C by peptidoglycan was measured.Whenpurified peptidoglycan was incubated for 30 minwith normal serum in concentrations of from 5.0 to 0.01mg/ml serum, a clear dose-response relationship wasestablished (Table II). When concentrations of 1.0-5.0mg were used, CH50 activity was not detectable.

Kinetics of C activationi in normal and C2-deficientsera. The ability of peptidoglycan to activate C in thepresence and in the absence of C2 was studied byincubating normal and C2-deficient sera with peptidog-lycan, at a concentration of 1.0 mg/ml, at 37°C. Sampleswere removed at 1-, 5-, 15-, 60-, and 120-min intervalsand centrifuged at 2°C. The hemolytic titer of C3-C9

TABLE IIComplement Activation in Normal Seruim by Peptidoglycatn *

Peptidoglycan CH,w

nig

5.0 <10t2.5 <101.0 <100.5 130.1 250.01 33Control 31

* 1-mi aliquots of normal serum were incubated with equalvolumes of purified peptidoglycan, in concenitrations from 5.0to 0.01 mg, and of water (control) for 30 min at 37°C. After ceni-trifuging, CH50 was measured in the supernatant serum.t <10 = not detectable.

was measured in the supernatant serum, and val-ues were compared with C3-C9 levels measuredin simultaneous controls (normal and C2-de-ficient sera incubated with water for the sametime intervals). Consumption of C3-C9 in thepeptidoglycan-treated samples was expressed as apercentage relative to the C3-C9 levels in the controls(Fig. 7). As shown, activation of comiiplemnent occurred1)oth in the presence (normal serum) and in the absence(C2-deficient serum) of an intact classical pathway;however, the rate of activation in C2-deficient serumwas significantly slower than in nornmal serum (Fig. 7).

100 _

( 80 Normal Serum

innrmal and C2-Deficient Serum

c600

CL

0

02

0

Minutes

FIGURE 7 Kinetics of C activation by puirified peptidogly-canin normal and C2-deficient sera. 1-miiI aliquots of normial andC2-deficient sera were incubated at 37°C with equal volumesof water (controls) and peptidoglx-can (1.0 mg) for 1, 5, 15, 60,and 120 min. Consumption of C3-C9 in the peptidoglycan-treated serum samples is expressed as a percent of thesimultaneous conitrol samples.

Opsonization of Staph ylococcal Peptidoglycan 6605

Effect of preabsorptiotn of serum with peptidoglycanlon subsequent C activation. The ability of peptido-glyean to activate the C system in normal serum wasfound to be significantly impaired when serum waspreabsorbed with peptidoglycan at 2°C. Although therewas 92, 89, and 37% conisumptioni of C3-C9 in controlserum after a 30-min incubationi with 5.0, 1.0, and 0(.1mg peptidoglycani, respectively, there was <25%C3-C9 conistuinption in serunm that had been preab-sorbed with peptidoglycan. Likewise, preabsorption ofC2-deficient serum with peptidoglycani at 2°C had aninhibitory effect on the subse(uent capacity of pep-tidoglycan to activate the C system. After 30-min and60-min incubations with 1.0 mg peptidoglycan, therewas 9 and 44% C3-C9 consumption in nonabsorbedC2-deficient serum compared with 0 and 27%activationin C2-deficient serum which had been preabsorbedwith peptidoglycan. The absorption procedure itselfdid not significantly influence C3-C9 levels (afterabsorption, C3-C9 levels were 422 and 540 U/ml innormal and C2-deficient sera compared with controlvalues of 437 and 500 U/ml, respectively). These datasuggested that peptidoglycan activation of the C sys-tem, via both the classical and alternative pathways,depended upon the presenice of an absorbable,nioncoimiplement-related serum factor.

DISCUSSION

The cell wall of most S. aureuis strains is composed ofthree major constituents -peptidoglycan, teichoicacid, and protein A. The peptidoglycan component is alinear polymer of repeating ,l-I -4-linked N-acetylgltu-cosamine and N-acetylmuramic acid residues. Attachedto the carboxyl group of each N-acetylmuramic acid is atetrapeptide cross-linked by a pentaglycine bridge to aneighboring peptide Unlit. Approximately 50% of theweight of the cell wall is contributed by peptidoglycan,and it is this component which provides the wall itsbasic rigidity (40). Cell wall teichoic acids, comprising-40% of cell wall weight, are charged polymers ofribitol-phosphate linked to muramic acid residues ofthe peptidoglycan (41). Protein A, contributing -5% tocell wall weight, is a protein with unique propertieswhich, in general, interfere with the process ofopsonization (42, 43). In this investigationi, trypsindigestioii was used to remove protein A and other cellNwall proteins. Also lipoteichoic acid, which may beanother comlponent of' the staphylococcal cell sturface(18), was extracted, and the two major constituenits ofthe cell wall -peptidoglycani and teichoic acid-werestuidied. The staphylococcal cell surface may be viewedas a mosaic of different dcomains where all of the majorcell wall components, inicluding peptidoglycan ancdteichoic acid, can be sufficienitly exposedl to interactwith external factors.

The relative importance of the peptidoglyean andteichoic acid coomponents of the S. aureus cell wall inthe process of opsonizationi has been a matter ofcontroversy. Evidence has previously been presentedsupporting the role of both teichoic acid (7-10) and(peptidoglyean (11, 12) in this regard. These studies,however, have used indirect methodls to assess opsonli-zation, either bN, immunn izinig aiiinials with cell wallcomponents and measuring the protective effect of'suchimmunizationi (8, 9) or by absorbing seruml with cellwall componients and determininig the effect of suchabsorption on serum opsonic activity (7, 11, 12).

In this study, PMNleukocyte uptake of intact S.aureus H and of highly purified cell walls-composedof peptidoglvcan and teichoic acid-and of isolatedlpeptidoglycan was measured quantitatively using[3H]glycine-labeled l)acteria, cell walls, and peptido-glycan. Whennormal human serum and heat-inactivatedlserum were used as opsoniic sources, intact bacteria,cell walls, and peptidloglvcan were takeni up by PMINleukocytes in a simlilar manner. All three particles weremost efficiently opsoniized bN normal serum, and uptakedid not occur in the absence of seruim factors. Electronmieroscope studies revealed that opson-iized peptido-glycan was readily ingested by PM1Nleukocytes. Studieswith purified IgG indicated that IgG was the miajor-heat-stable serumni opsonic factor, a finding reporte(d byother investigators (44-46) for different staphylococcalstrains.

As isolated cell wNalls (peptidoglycan and teichoicacid) and purified peptidoglycan were phagoeytized ina comparable fashioin, it appeared that the remtoval ofteichoic aci(l fromii the cell wvall did not significantlyaffect opsonization, thereby implying that the processof opsonization wvas primarily in ediated by pepti doglv-cani. Electroni microscope stuidies, however, had re-vealed that the cell wall anid peptidoglvcan prepara-tions were nlot morphologically identical; in thinisections the peptidoglycani was, in generall, imiorecollapsed. Therefore, the possibility existed that thisstructural difference might inifluience opsoniizationi in amanner independently of the well-defined chemiiicaldifference of these particles. To help clarify thispossibility, opsonization of' a teichoic acid-deficienitmultanit straini of S. auireuis H was compared with that ofthe parent strain. The abseniee of teichoic acid ini thecell wall di(l not alter opsoniizationi by niormiial and(heat-inactivated sera which aclded support to thehypothesis that teichoic acid played anl insignificantrole in bacterial opsonizationi.

The findinig that there was no PMNI leukocyte uptakeof lysostaphin-degraded peptidoglycan which had beenpreincubated with normal and heat-iniactivated serastuggested that a certain particle size is re(Itqired foreffective opsonizationi. Shavegani and colleagues (11)reported similar findings using differen)t mIethods anid

606 Petersoni, Wilkinl.soni, Kiml, Verhoef, Schmelintg, Douglas, and ()uie

enzvymes. Grov and co-workers (47) have recentlydemonstrated that the iihibition of leukocyte migrationby staphylococcal peptidoglycan is also dependentupon the size of the peptidoglycan fragmiient.

Other investigators have demonstrated that theclassical (46, 48) and the alternative (46, 49, 50) Cpathways are involved in staphylococcal opsonizationi.In anl attempt to delineate which heat-labile serumfactors participated in the opsonization of S. aureus Hand of the peptidoglycan comiiponent, phagocytosis ofintact bacteria, cell walls, and peptidoglycan wasmeasured after opsonization with normal, MgEGTA-chelated, heat-inactivated, C2-deficient, and heat-inactivated C2-deficient sera. Opsonization of all threeparticles was optimal when normal serum was used asan opsonlic source. In the absence of C2, opsonizationwas less effective, both the opsonic capacity aind therate of opsonization being less with C2-deficient serumilthan with normal sertum; however, opsonization didproceed via a heat-labile system (the opsonic capacity ofC2-deficient serum being significantly, reduced byheat-inactivation). On the other hand, heat-labileopsonic activity vas al)olished in the absence of Cl(MgEGTA-chelated serumiii).

These results suggest that opsonization of intact S.aureuis H anid of peptidoglycan is most effective in thepresenice of an intact classical pathway. However, itappears that opsonization can proceed via anotherheat-labile pathway when C2 is blocked but not wheniCl is blocked. Other investigators (51, 52) havedemonstrated that C1 can play a role in the activation ofthe alternative complemiient pathway (the "Cl-bypass"mechanism). From the results in this study, it appearsthat the Cl-bypass mav be operative in the opsonizationof S. atureus H peptidoglycani by C2-deficient serum,and that blockiing Cl thereby interferes with alternativepathway activation.

Activationi of the serumii C system by peptidoglvcanwas further studied bv incubating peptidoglycan withnormal serumiii anid miieasuring C constumption. A (lose-response relationiship was established in which CH5,was not detectable after a 30-mmliil incubation withpeptidoglvycan in conicentrationis of 1.0-5.0 mg/ml seninm.The kinetics of C activation were then investigatedlusinlg normlal an(l C2-deficieint sera. Although the rate ofC3-C9 coinstumiiptioni Vwcas significanfly faster and pro-ceede(d to a greater degree in niormiial serumii, C3-C9 wasalso consumiied in C2-deficient serumii. These findingswere consistenit with observations miade in the funic-tionIal phagocytosis assay whereinl C2-deficient seruimlwas nlot as effective as normiial serum as an Opsfoiicsource aiid opsonlizatioin proceeded at a slower rate inC2-deficient serumni than in niornmal serumiii. Simiiilarfindinigs have beeni reported in studies of opsoniizationiof other staphylococcal strainis (28, 42). Recentl-,Bokisch (53) has reported that peptidoglycans froml

Streptococcus group A, Staphylococcus epidermidis,and Micrococcus lysodeikticus were capable of activat-ing both the classical and the alternative C pathways inhumnan seruim. Pryjma and colleagues (4) have demon-strated the same capacity for S. aureus peptidoglycanusinig serumil from guinea pigs and mice.

In anl attempt to determine whether S. aureuspeptidoglycan was capable of directly activating theclassical and alternative C pathways or whether Cactivation was initiated by antigen-antibody complexformation, normal and C2-deficient sera were preab-sorbed with peptidoglycan at 20C before addingpeptidoglycan at 37°C and measuring C3-C9 consump-tion. Preabsorption of both serum sources with pep-tidoglyean was found to limit the ability of peptidogly-can to subsequently activate the classical and alterna-tive pathways of C. These preliminary findings suggestthat C activation by peptidoglycan depends upon thepresence of an absorbable, noncomplement-relatedfactor, presumnabl im munoglobulin. Wheat and co-workers (54) have presented data to stupport this hypothe-sis. Activation of the classical C pathway, in general, isknown to be initiated by the combining of IgG or IgNMwith antigen (55). There is a growing body of evidencewhich suggests that, in certain systems, activation of thealternative complement pathway may likewise dependuiponl antigen-antibody formation (46, 56, 57). An-tibodies of the IgG class, to S. aureus peptidoglyeanhave been showin to be present in normal human serum(58). However, there is a need for studies utilizingpurified C componeints to evaluate the possibility thatpeptidoglycain may directly interact with Cl and/ordirectly activate the alternative C pathway as describedfor certain gram-negative bacterial cell wall compo-nieints (59).

Over the past decade it has been demonstrated thatthe recognition of staphylococci by PMNleukocytes ismediated by receptors within the phagocytic mem-brane with specificity for the Fc fragment of IgG and foranl activated form of the third component of C, C3b (60,61). Staphylococcal peptidoglycan would appear to playa ceentral role in the initiation of this recognitionprocess. Other investigators (1, 62) have proposed thatthe peptidoglycan, a maijor cell wall component of mostgranm-positive bacteria, may play a significant role in thedevelopmenit of' "natural immunity" to this grouip ofmiieroorganisms. Recognition of all gram-positive bac-teria may, therefore, be a manifestation of host responseto this comminioni cell wall conmponent.

It shouild be emphasized that although cell wallteichoic acicl was found not to be important inpromlotinig opsonization in the serunm sources used inthis stuLdyl, the possibility remains that teichoic acid is ofimlportance in fostering opsonization in serunm froImlpatienits with S. auireus iinfections in which inereasedlevels of teichoic acid anitibodies have been demnon-

Opson ization of Staphyllococcal Peptidoglycan 6607

strate(d (63). Futuire stu(lies vill be directed at inves-tigatinig this possibility.

ACKNOWLEl)G MENTS

'I'l)c auitlhors aire gratefull to D. Petersoni f'or the aIIiii0 acidanalyses, to Patti Lorenz f'or her secretarial assistancie, anid to L.D. Sabath for hiis valuable discuissioni aind suipport.

This work wasstupportecl hv U. S. Public Health Servicegraniits Al 06931-11, AI-08821-07, Al 12478-03, Al 10704, anidIIL 06314; a granit fromii the Kroe Founda(ltioni; and aGrant-in-Aid f'romii the Graduate Sehool, University of Minz-nesotat.

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