The key role of peptidoglycan in the opsonization 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.

Research Article

In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of 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 the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that 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 the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host […]

Find the latest version: https://jci.me/108971/pdf The Key Role of Peptidoglycan in the Opsonization of Staphylococcus Aureus

PHILLIP K. PETERSON, BRIAN J. WILKINSON, YOUNGKI KIM, DAVID SCHMELING, STENVEN D. DOUGLAS, and PAUL G. 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 R A C T In an effort to determine the staphylo- INTRODUCTION coccal cell surface component(s) of importance in opsoniization, cell walls (peptidoglycan and teichoic In recent years increased attention has focused on the acid) and peptidoglycan were isolated from Staphylo- biological significance ofthe cell walls of gram-positive coccus aureus strain H grown in [3H]glycine-con- bacteria. In particular, peptidoglycan, a major compo- taining broth. After incubation of the cell walls nent of the cell wall of most gram-positive bacteria, has and peptidoglycan with various opsonic sources, been shown to play a significant role in determining the uptake by human pol'ymorphonuclear leukocytes nature of host response to this group of bacteria (1, 2). In wvas measured. The opsonic requirements for phago- the case of Staphylococcus aureus, this cell wall cvtosis of cell walls and peptidoglycan were found component has been demonstrated to possess to be similar to those of intact bacteria. Removal endotoxin-like properties (3), to activate the serum of teichoic acid from the cell wall did not affect complement (C) system (4), and to elicit a cell-mediated opsonization. Likewise, a teichoic acid-deficient mut- immune response (5, 6). Few studies, however, have ant strain of S. aureus H was opsonized in a manner examined the role of the cell wall components of S. similar to that of the parent strain. Immunoglobulin G aureus in the process of opsonization, and results have functionied as the major heat-stable opsonic factor and been conflicting regarding the relative importance of both the classical and alternative pathways participated these components in this process (7-12). in opsonization. Kinetic studies revealed that opsoniza- In this investigation the role of the two major tion of peptidoglycan, as well as C3-C9 consumption components of the S. aureus cell wall-the peptidogly- by peptidoglycan, proceeded at a slower rate via the can and teichoic acid-in the process of opsonization alternative pathway (C2-deficient serum) than when was studied by measuring the uptake of radioactively the classical pathway was present (normal serum). The labeled bacteria and purified cell wall components by ability of peptidoglycan to activate C3-C9 was sig- human polymorphonuclear (PMIN)' leukocytes. The nificantly reduced when normal and C2-deficient sera nature of the opsonic factors involved in this process were preabsorbed with peptidoglycan at 2°C suggest- and the kinetics of C activation were examined. Data is ing that antibodies to peptidoglycan may be involved in presented which demonstrates that opsonization by activation of'both the classical aind alternative comple- both heat-stable (IgG) and heat-labile serum factors is menit pathways. Thus, peptidoglvcan appears to be the mediated by cell wall peptidoglycan. Peptidoglycan kev cell wall comiipoineint iinvolved in staphylococcal was found capable of activating both the classical and opsonlizationi, aind it is suggested that host response to alternative pathways ofthe C system. It is proposed that peptidlogl-can, a major cell wall component of most Cl may' play a role in activation of the alternative gramn-positive bacteria, max be related to the develop- comiiplenmeint pathway by peptidoglycan and that activa- miienit of i'natniral imm-niunity" to this group of mnicroor- tion of both the classical aind alternative pathways is ganlism11s. initiated by antigen-antibody comiiplex formation.

Dr. Qoiie is the Aier-icani Legion Memorial Heart Research Abbreviatiotis utsed in this pa per: CH5(,, total liemol tic Professor. compleim1ent; HBSS, Han1ks' balaiced salt solutioin with 1% Rece ied for publication 4 August 1977 aoid in revi.sedfor1;1 gelatini; PBS, phosphate-buffered saline; P.MN, polvmor- 27 October 1977. phon icleair.

The Jouirtnal of Clintical Itncestigation Volutmne 61 Marchi 1978 597-60(9 597 METHODS comlponents. The cell walls were then washed four times in water at roomii temperature before being resuspended in 200 ml Bacterial strains 0.05 M Tris-HCI containing MgCI2 (5 mM) plus 2.5 ,ug/ml each of RNase (EC 3.1.3.4, 2,588 U/mg) and DNase (EC S. aureus strain H, choseen because of its well-estal)lished 3.1.4.22,2,500 U/mg) (each type 1 from Worthington Biochem- cell wall structure (13), was kindly provided by Sir James ical Corp., Freehold, N.J.) and incubated at 37°C for 1 h with Baddiley's laboratory, University of Newcastle-upon-Tyne, gentle shakinig (100 rpm). To remove proteinaceous material England. S. aureus HSImIR (a spontaneous streptomycin- including protein A (17), 200 ,ug/ml, trypsin was added (EC resistant isolate of strain H) and a teichoic acid-deficienit 3.4.4.4, 189 U/ml, Worthington Biochemical Corp.), and the mutant, straini 52A5 (14), were generously provided by J. T. incubationi contintued for 4 h. The walls were washed once in Park, Tufts University, Boston, Mass. The cell wall of strain water and then stirred with an equal volume of 80% phenol for 52A5 appears to be composed almost entirely of peptidoglycan 30 lnin at room temperatuire to remove lipoteichoic acid (18). (15). The organisnms were maintained on Trypticase Soy Agar After extraction, the walls were washed six times in water and (Difco Laboratories, Detroit, Mich.) slants. then lyophilized. 1,680 mg of cell wall was recovered from 26 liters of culture. Cell walls (1,260 mg) suspended in 75 ml water were mixed Cultivatiotn and radioactive labeling of with 75 ml of 20% (wt/vol) TCA and were incubated at 60°C bacteria for 90 min to remove teichoic acid (19). The suspenision was cenitrifuged to yield peptidoglyean which was washed four Bacteria were grown in peptone yeast extract broth (16) with times with water before being lyophilized (yield 1,067 mng, aeration on an orbital shaker at 37°C. For phagocytosis studies 35.5% weight of wall removed.) Teichoic acid was recovered of intact bacteria, the organisms were labeled by inocuilatinig from the suiperniate by adding 750 ml cold EtOH and allowing several coloniies of bacteria into 20 nml peptone yeast extract to stand at 4°C overnight. The precipitate recovered by broth containing 40 ,uCi [2-3H]glycine (sp act 5-15 Ci/mmol, centrifugation was resuspenided in 14 ml 10% TCA and New England Nuclear, Boston, Mass.). After an 18-h incubation allowed to precipitate again overnight at 4°C with 70 ml EtOH. at 37°C, the bacteria were washed three times in phosphate- The product was washed twice with acetone and allowed to air b6uffered saline (PBS), pH 7.4, and resuspended in PBS to a dry. final concentration of 1 x 109 colony-forming units/ml using a Typically, cell walls were labeled to a specific activity of spectrophotometric method confirmed by pour plate colony -5 x 105 cpiml/milg dry weight. The hot TCA treatment resulted counts. For preparing radiolabeled cell walls, organisms were in about 10% loss of counts indicating little solubilization of grown in peptone yeast extract broth, 2 liters containing glycine bridges occurred (20) and the specific activity was [2-3H]glycine, 0.4 ,uCi/ml, and were harvested in late roughly dcouble that of the cell walls. Preparations were stored exponential growth phase (OD625 nm 1.5-2.0, Spectronic frozen at - 15°C at a concentration of 5-10 mg dry weight/nil, 20, Bausch & Lomb Scientific Optical Products Div., Roches- and brief treatment in a soInic bath (Branson Sonic Power Co., ter, N.Y.). Large scale growth of unlabeled bacteria (26 liters) Danbury, Conn.) was used to help attain an even suspension was achieved in a New Brunswick Fermentor (New Brunswick before each opsonization procedure. Scientific Co., Inc., New Brunswick, N.J.) at 370C with forced aeration (5 liters/min) usinlg anitifoam (Anti-Foamii aerosol spray, Sigimia Chemical Co., St. Louis, Mo.). 2 liters of a 15-h Chemical characterization of cell walls, culture was used as an inoculum, and the bacteria were grown peptidoglycan, and teichoic acid for 6-7 h and harvested at an OD625 nm of 1.0-1.5. The cultures were harvested in a continuous flow centrifuge Quantitative examination. Carefully weighed cell walls, (Beckman J-21) using a JCF-2 rotor (Beckman Instruments peptidoglycan, and teichoic acid were hydrolyzed in sealed Inc., Spinco Div., Palo Alto, Calif:) operating at 2,500 g with a tubes with 6 M HCI for 18 h at 105°C. Acid was removed under flow rate of 250 ml/min. The organisms were washed once in vacuum over anhydrous CaCl2 in the presence of NaOH cold distilled water and then frozen (- 15°C) overnight before pellets; the dried residues were twice moistened with 0.5 ml disruption. The yield was 80 g wet weight of cells from 26 water and redried. Amino acids and amino sugars were liters of culture. estimated on an aminio acid analyzer (Beckman model 120B Amino Acid Analyzer, Beckman Instruments, Fullerton, Calif. Inc.,). Phosphoruis was estimated by the method of Fiske and Isolation of cell walls, peptidoglycan, and Subba Row as described by Leloir and Gardini (21). Total fatty teichoic acid acids in 10-mg amounts of preparation were determined as described by Wilkinson (22). Unless otherwise indicated, all operations were carried out Qualitative examination. Teichoic acid (5 mg) was at 40C and all centrifugations were at 16,319 g for 10 min at 40C. hydrolyzed in 2 M HCI for 3 h at 105°C in sealed tubes; acid Washed organisms in distilled water were mixed with glass was removed as described above. Part of the hydrolysate was beads (0.10-0.11 mm, VWR Scientific Div., UNIVAR, San treated with alkaline phosphatase (EC 3.1.3.1, 1 mg, type 1, Francisco, Calif.), and disrupted by agitation in a Vibrogen calf intestinal mucosa, Sigma Chemical Co.) in 0.025 M Cell Mill (R.H.O. Scientific Inc., Commack, N. Y.) for 20 min at NH4HCO3 containing 0.005 M NH40H, pH 8.3, over- maximum speed. The glass beads were removed by filtration night at 37°C. Alkaline phosphatase-treated and untreated on a coarse glass sinter and the filtrate was centrifuged to hydrolysates were examined by descending chromatog- deposit the white cell walls overlaying yellower, unbroken raphy on Whatman Ino. 1 paper in the solvent systems 6 organisms. Care was taken to avoid resuspending unbroken propan-1-01:3 0.88 NH3: 1 H,O (by volume) and 6 butan-1-01:4 organisms. The crude cell walls were washed once in distilled pyridine: 3 H20 (by volume) (23). Reducing compounds were water, resuspended in 200 ml 2% sodium dodecyl sulphate, detected with the alkaline AgNO3 reagent (24) and compounds and then stirred overnight at room temperature to inactivate with amino groups by ninhydin treatment (0.25% wt/vol in autolytic enzymes and to aid in stripping of membrane acetone plus heating at 100°C for 5 min).

598 Peterson, Wilkinson, Kim, Verhoef, Schmeling, Douglas, and Quie Opsonic soU rces PMN leukocyte suspension was added. These mixttures were incubated at 37°C in an incubator shaker (New Brunlswick Serum was pooled from five healthy donors who cleniied Scientific Co., Inc.) at 250 rpm. PMN leukocyte uptake of previous staphylococcal infection (normnal serum). To block bacteria, cell walls, and peptidoglycan was quantitated using a C I, normal serumn was chelated with ethylene glycotetraacetic modificationi of a previously described method (28). Briefly, acid in the presence of MgCI2 (MgEGTA) in a 10-M to 10-mM leukocyte-associated radioactivity was determined by taking concentration with respect to serum (25). C2-deficient seruml] duplicate 100-,ul samples from each phagocytosis mixture at was obtained froml a patient with genetically determined specified time intervals, placing the samples in 3 ml PBS, pH complete albsence of C2 and normal levels of all other C 7.4, in polypropylene vials (Bio-vials, Beckmiiani Instrumnenits comlponents. Serum was stored in 0.5-mll ali(luots at -70°C. Inc.,) and washing the leukocytes three times in PBS by Heat-iniactivated serum was prepared by heating thawed differential centrifugation at 160 g. Final pellets were resus- aliqtuots of serumil at 56°C for 30 mni. Purified humani IgG was pended in 3 ml scintillation li(quid (toluenie containinlg provicled bh Dr. D. Roos, Central Laboratory of the Blood fluorallov [TLA, Beckman Instruments, Inc.] and 20% Transfusion Service, Amsterdanm, Netherlanids. Serum and Biosolv-3 [Beckman Instrunmenits, Inc.]) and couniited in a IgG were diluted to specified coneentrations with Hank's liquid scintillation counter (Beckmain LS-250). To determine balanced salt solution conitaining 1% gelatin (HBSS) just total radioactivity (representing both leukocyte-associated aold before use. Calciumil- andl magnesium-free HBSS was usedl to nonleukocvte-associated bacteria, cell walls, or peptidoglv- (lilute M\gEGTA-chelated serum. cai), duplicate lO0-pl samples were placed in 3 ml PBS at the end of the assay period and centrifuged at 4,000 g for 15 min. Supernates were discarded, pellets resuspended in scintilla- Opsonization procedure tion liquid, and samples counted in the scintillation counter. An According to Chatterjee (26), _1 x 108 whole organismiis average of duplicate values was used in all calculationis. PMN yield 25 Ag of cell walls. Conise(luently, 0.1-mI ali(quots of leukocyte uptake of radioactively labeled bacteria, cell walls, intact bacteria (1 x 1)8 colony-forming units) and 25 lig of cell and peptidoglveacni at given sanmpling times was calculated walls were used in all opsonizationi experimiienits. Studies tisiig using the formula: %N uptake = (cpm in leukocyte pellet)/(total 2.5 and 12.5 jg of peptidoglycan yielded similar find(lilgs; cpmli) x 100. results were reported using 25 Ag of peptidoglycan. Each To study opsoniization of soluble peptidoglycan fragnmenits, aliquot was incubated at 37°C in a 12 x 75-mm plastic tube, purified peptidoglycan was digested with the muralvtic BioQtuest, BBL & Falcon Products, Becton, Dickinson & Co., enzyme, lysostaphin. 0.5 mg (0.1 ml) peptidoglycan wvas Cockeysville, Md., containing 1.0 ml of the indicated opsonlic inicubatedl in 0.9 ml PBS containing 10 gg/ml lvsostaphini source for specified tinmes followed by centrifuiginig at 4,000 g (Schwartz Mann Div., Becton, Dickinson & Co., Orangeburg, for 15 min (4°C). The supernate was discarded and the pellet N.Y.) for 60 min at 370C followed by centrifuging at 10,000 g for resuspeinded in 0.5 ml HBSS. 30 min. Suipernates were then heated in a boiling water bath for 15 min to inactivate lysostaphin. 50-,ul aliqIuots of the stipernate (peptidoglycan fragments) were then incubated with opsonlic PMN leukocytes sources for 15 min. PMN leukocytes were added, and uptake Blood from healthy donors was drawni into heparinlized wvas determined as described above. Total radioactivity in syringes (10 U heparin/ml blood). PMIN leukocytes were these mixtures was determined by suspendinig 100-,ul salmples prepared using a method modified from B16yum (27) in which directly into sciintillation liqiuid. A control mixtuire of 0.5 mg the blood was centrifuged for 15 mmi at 200 g, and the peptidoglycan was incubated in PBS for 60 min; after plasimia-platelet layer was removecd with a Pasteur pipette. The centriftiging, the supernate was discarded and the pellet leukocvte layer was then withdrawn, placed in plastic tubes, resuspenided in 1.0 ml PBS containing 10 ug/ml lysostaphini and then diluted with two parts Spinner's Minimal Essenitial which had been previously heat-inactivated. This mixture was Medium (iX, Grand Island Biological Co., Grand Island, N. Y.). then heated in a boiling water bath after which 50-,l ali(quots 8 ml of this suispenisioni was carefully layered onto 3 ml were removed for opsonization and phagocytosis studies. Ficoll-Isopaque (lymphocyte separation medium, Litton Bionetics, Kensinigton, Md.). After centrifuging at 200 g for 30 Measuremnent of C activation by purified mmin the mononuclear cell laver was removed with a Pasteur pipette. To obtain pure PMN leukocytes, the remaining peptidoglycan medium was withdrawni and discarded, and 0.87% NH4Cl in 0.1-ml ali(qtuots of peptidoglycani, at specified conicenitra- sterile water was added to the tube containing the tions, and of water (control) were added to 12 x 75-mm erythrocyte-PMN leukocyte pellet which was then rotated for plastic tubes (BioQuest, BBL & Falcon Products) conl- 10 min at room temperature. After centrifuginig for 10 min at taining 1.0( ml normal serum. After a 30-mmi incubation at 160 g, the PMN leukocyte pellet was washed an additional two 37°C, the tubes were centrifuged at 4,000 g for 15 min (4°C) and times with 0.87% NH4CI to lyse the renmaining erythrocytes. supernates were stored at -700C before measurement of total The PMN leukocytes were resuspended finally in HBSS and hemolytic complement (CH50). total and differential leukocyte counts were performed. The To study the kinetics of C activation in normal serum and in final leukocyte pellets were adjusted to a concentration of 1(7 C2-deficient serum, 0.1-ml (1.0 mg) aliquots of peptidoglvcan PMN leukocytes/ml HBSS. Contamination by mononuclear were added to 1.0-ml samples of serumn followed by incubating cells was evaluiated by Wright's stainied smears and generally at 37°C for indicated time intervals. Control mixtures did niot exceed 1%. containing 0.1 ml distilled water were incubated simultane- ously and removed at the same time intervals. After cenitrifug- Phagocytosis mixtures and assays ing at 4,000 g for 15 min (4°C), the supernates were stored at -70°C before measurement of C3-C9. To each 0.5 ml suspension of opsonized radioactivelv The effect of preabsorption of normal and C2-deficient sera labeled intact bacteria, cell walls, and peptidoglycan, 0.5 ml of with peptidoglyean on the subsequent ability ofpeptidoglycan

Opsonization of Staphylococcal Peptidoglycan 599 to activate the serum C system was studied by incubating TABLE I 1.0-ml sami-ples of serum three times for 60 min at 2°C with Chenmistry of Cell Waoll.s, Peptidoglicat, (al)1 'Tichoic Acid8 either 0.1-ml (1.(0 mg) aliquots of peptidoglycan or 0.1-mil aliquots of distilled water (nonabsorbed serum). After each Conlipolnelnt Cell walls Peptidoglycand Teicloic acid 60-min absorptioni period, samples were centrifuged for 15 min at 4,000 g (2°C) and the supernatant serum was added to the next precooled tube conitaininig either peptidoglycan or water. After the final centrifugation, the serum samples were Muramic acid 180.4 280 Trae added to tubes containing 0.1-ml aliquots of peptidoglycan, at Glucosanmine 682 412 1,560 indicated concentrations, and were then incubated at 37°C for Glutanmie acid 277 668 24 specified time intervals. Control tubes containing absorbed Lysine 444 812 48 and nonabsorbed serum and 0.1 Iml distilledl water were Alanine 849 1,588 :310 likewise incubated at 37°C. After incubation, samples were Glycine 1,826 3,460 120 centrifuged at 4,000 g for 15 min (2°C) and the supernates stored at -70°C before C3-C9 determination. Seriine 48 68 - Hemolytic titrations of C were performed using sheep Phosphate 1,((( 84 2,000 erythrocytes in acid citrate dextrose (Wilfer Laboratories, Aspartic acid 26.4 24 Stillwater, Minn.). Intermediate cell preparation (C14) and the Threonine 8.8 8 -- assays of CH50 and C3-C9 were performed as previously Valine 8.8 8 described (29-32). For CH50, the optical density was read at Isoleucine 8.8 8 541 nm and for C3-C9 titration at 412 nm. Results were Leucine 8.8 8 expressed as 50% lysis, and titrations were corrected for Histidiine 30.8 20 day-to-day variations by including a knowin internal standard Arginine 8.8 16 human serum. * Cell walls, peptidoglINcain, and teichoic acid wvere isolatedl Electron microscopylJ from S. aureus strain H. Cell walls and peptidoglycan were examined by a Siemens 102 electron microscope (Siemens Corp., Medical/Industrial Groups, Istlin, N. J.) using previously described methods (33). ponents. Assumiiing that ribitol phosphate and N-acetyl- In addition, 0.1-ml aliquots of peptidoglycan (1 mg) were glucosamine are equimolar then one can take ribitol, incubated in plastic tubes (BioQuest, BBL & Falcon Products) 1.0; phosphate, 1.0; N-acetylglucosamine, 1.0; containing 1.0 ml 10% normal serum, 10% heat-inactivated alanine, serum, and HBSS for 60 min. After centrifuging for 15 min at 0.16 as the average teichoic acid sub-Un1it (see lelow) 4,000 g, supernates were discarded and 0.5 ml HBSS was added and this gives a formula weight of 410. Thus, 41% of the to each tube. Leukocyte suspensions (0.5 ml) were then added dry weight of the cell wall is present as teichoic acid and the mixtures incubated for 15 min at 37°C. The leukocytes plus 50.5% as peptiodglycan thereby accounting for were washed three times with cold PBS, fixed in 1.5% glutaraldehyde in PBS, and then thini sections were examined. 91.5% of the dry weight of the cell wall. It is likely that metal counterions to negatively/ charged groups in the teichoic acid and peptidoglycan mlake up a RESULTS considerable portion of the uinaccounited wveight of Chemistry of the cell walls, peptidoglycan, and these preparations (35). 82% of the teichoic acid teichoic acid preparation is accountable for as teichoic acidl componients. Table I shows the quantitative analysis for the cell Additional evidence that the peptidoglycan is free of wall preparations used in this study. It can be seen that teichoic acid is the finding that 95% of the cell wall amino acids other than those normally considered to be phosphorus was renmoved by the hot TCA extraction andl part of the staphylococcal peptidoglycan were either the ratio of the glucosamine to muramic acid chaniged not detected or present in only small amouints. The lack from an excess to being almost e(uimiiolar. of the full range of protein amino acids indicates little Qualitative examination of the teichoic acid hydroly- contamination of the cell walls with proteinaceous sate revealed components with chromatographic m11o- materials including protein A. On the basis of the bilities characteristic of glucosamiiiie, ribitol, altnine, analyses presented here, and knowledge of the S. glucosaiminitol phosphaite, ribitol di- aiud inoniophos- aureus peptidoglyean structure (19, 34), it can be seen phate, and glucosaminitol ribitol. Amino acid analyses that the average subunit of the peptidoglycain containis of the teichoic acid revealed glticosamine anld alanine isoglutanmine, 1.0; lysine, 1.0; alanine, 2.0; glycine, 4.0; to be the imnajor componients. The al)sence of miiramnic serine, 0.1; N-acetylmuramic acid, 1.0; N-acetylglucos- acid andl the low amounts of other a-n)ino iacids show amine, 1.0; O-cacetyl, 0.6; aind the formula Nweight that the teichoic acid fratctioni does nlot conitaii majorn is 1,138. Using the lysine value it cani be seen amounts of peptidoglyca.n In .an effort to (leteit-rinc that 50.5% of the dry weight of the wall is w,hether the cell wvall aind peptidoglycan pre piar.tions accouinted for by the peptidoglycan. For isolated were contami-niated with lipoteichoic acid, the total peptidoglycan, 92.4% of the dry weight of the f:atty ateid comiipositioni of the prepiarations \wTajS preparation is accountal)le for as peptidoglycan com- dleternii ined. Fatty atci(ls ar-e imarkers of eel1 ineihl)ran e 600 Peterson, Wilkin son, Kitl, Verhoef, Sclhlneling, Douglas, andlI (Jo ie 4mohn

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FIGURE 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 with or enzvnmes but had simply been washed six times in the fattv acid compositioni of"crude cell walls" that had water. Five major fatty acid methvl ester peaks were not been treated with sodiumil dodecvl sulfate, phenol, obtained from the crude cell walls by gas-liquid Opson ization of Staphylococcal Peptidoglycan 6016 chromatography. Chromatography of the fatty acid 100_ methyl ester fraction from an equal weight of the in study revealed only one purified cell walls used this 80 _ very small "peak" on the trace that was difficult to .cw estinmate. Similar examination of the peptidoglycan it.orNS revealed two extremely small peaks. Summation of the 60 total detector response revealed that the cell walls and peptidoglycan had at least 97.4 and 98.7%, respectively 40 - less fatty acids than the crude cell walls. The cell wall iOO S.our anid peptidoglycan preparationis were contaminated PG AS with fattv acids at levels < 1 nmol/mg. These results, 20 -c the fact that we cani account for most of the weight plus Sour of the preparations in known conmponents as well as the cw HBSS proveni value of sodium dodecyl sulfate and phenol 0 3 10 15 extractions in renioving lipoteichoic acid (18, 36), Minutes indicate the preparations are not significantly con- FIGURE 2 PMN leukocyte uptake of intact S. aureus H (S. taminated with lipoteichoic acid. aur), isolated cell walls (CW) and purified peptidoglyean (PG) Thus, the qualitative and (uantitative analyses preincubated with 1% normal serum (NS), 1% heat-inactivated serum (AS) and Hanks' balanced salt solution (HBSS) before indicate that the cell walls (composed of peptidoglycan adding leukocytes and determining uptake at 3, 10, and 15 and teichoic acid) and peptidoglycan used in this min. Results represent means of three experiments. investigation are highly purified and chemically consis- tent with the observations of other investigators (15, 34). Electron micrographs of glutaraldehyde-osmium tetroxide-fixed, embedded sections of cell walls and can, electron microscope examination was performed peptidoglycan are shown in Fig. 1. The preparations are (Fig. 3). After opsonization with normal serum, pep- (luite homogenous and devoid of whole organisms and tidoglycan was present within phagocytic vacuoles in debris. Both cell walls and peptidoglycan reveal a the leukocytes and significant degranulation has oc- characteristic trilaminar appearance and septa (37). The curred. When preincubated with HBSS, essentially no morphologic appearance of the peptidoglycan is some- peptidoglycan was found within the leukocytes. Op- what collapsed and folded in comparisoni to the cell sonization with heat-inactivated serum resulted in less walls. In general, the peptidoglycan is thinner in ingestion than when normal serum was used as an sectionied material than the cell walls, a finding in opsonic source but in greater ingestion than when no agreemeint with previous studies (37, 38); detailed opsonin was present (HBSS). morphometric measuirements, however, were not per- Opsonic titer of normal and heat-inactivated sera. formed. When 1% serum was used as an opsonic source PMN leukocyte uptake of cell walls (peptidoglycan with Phlagocytosis of S. aureus H and of isolated cell teichoic acid) and peptidoglycan was found to be and similar. This result, as stated above, suggested that walls peptidoglycan removal of teichoic acid from the cell wall did not Kinetics of uptake by PMN leukocytes. Intact S. signifiantly affect opsonization. To test the possibility aureus H, isolated cell walls (peptidoglycan with that removal of teichoic acid might change the titer of covalently linked teichoic acid) and purified serum capable of effective opsonization, intact bacteria, peptidoglycan were incubated with 1% normal serum, cell walls, and peptidoglycan were incubated for 15 min 1% heat-iniactivated serum, and HBSS for 15 min before with varying concentrations of normal serum and adding PMNN leukocytes. As shown in Fig. 2, uptake of heat-inactivated serum before adding leukocytes and intact bacteria, cell walls, and peptidoglycan was determining uptake at 15 min (Fig. 4). Phagocytosis of simiiilar at 3, 10, and 15 min. At 15 min,-70% of each of' all three particles was significantly reduced and to the the three types of"particles" opsonized with normal serum same extent when 0.1% normal serum and when 1.0% were letkocyte-assoc iated compared with _25% and heat-inactivated serum were used as opsonic sources virtually nlo uptake of particles which had been thereby demonstrating that the opsonic titer was not incubated with heat-inactivated sertum and HBSS, influeniced by the removal of teichoic acid from the cell respectively. These restults stuggested that removal of wall. When purified human IgG, in concentrations of teichoic acid fronm the cell wall preparations did not 2.5, 1.0, 0.1, and 0.01 mg/ml, was used as an opsonic sign ificantly alter opsonizationi aind phagocytosis by source, uptake was found to be similar to that of 5.0, 1.0, PMN leuikocytes. 0.1, and 0.01% heat-iniactivated serum, respectively To determine whether levikocyte-associated radioac- (data not shown). This suggested that IgG was the major tivity represented attached and/or inigested peptidogly- opsonic factor in heat-inactivated serum and implies 602 Petersoni, Wilkin.son, Kim, Verhoef, Schmeling, Douglas, a(nd Quie 0 :- 4. 0 ib - .0 -1.7 .PfN- 1 ..4"m 11 -, .1.f!V 1. 4 -1. .K,

eA ir- IA.I e- ".I 4h i", . _::;-- A -%. 1, 'Wrol:. ,,-F

't. 1-

FIGURE 3 Electron micrographs of peptidoglyean after a 15-min incubation with PMN leukocytes. Peptidoglycan preincubated with HBSS remains extracellular, (a) x7,500; peptido- glycan preincubated with normal serum is present within phagocytic vacuoles and degranulation has occurred, (b) x7,500, (c) x50,OOO. that purified human IgG contains antibodies against though the results of the above experiments suggested peptidoglycan. that peptidoglyean was the major component involved Phagocytosis of S. aureus HSmR and 52A5. Al- in opsonization by normal and heat-inactivated sera, Opsonization of Staphylococcal Peptidoglycan 603 acid-deficient mutant did not differ significantly from that of the parent strain when varying concentrations of I)80 * S.aoureus H normal and heat-inactivated sera were used as opsonic 0 Ce/f Wo/ls sources. This result provided further evidence that the c~~~~~~~~~~ ~~~Pep/ldoglycan teichoic acid component of the cell wall did not have a E 60 major influence on bacterial opsonization. After each experiment the typeability of the strains was checked 4)40 with phage 52A (39). Teichoic acid forms part of the receptor site for this phage (14). The parent organism remained susceptible to this phage and strain 52A5 was 0 not susceptible, thereby inidicating that reversion of the 52A5 strain to a teichoic acid-containing form had not 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 occurred. Serum Concentration (%) The role of Cl and C2 in opsonizationi. To study the FIGuRE 4 PMN leukocyte uptake at 15 min of intact S. role of the classical C pathway in opsonization, Cl was aureus H, isolated cell walls and purified peptidoglycan blocked by chelating normal serum with MgEGTA, and opsonized with varying concentrations of normal serum (A) opsonization in the absence of C2 was studied by using and heat-inactivated serum (B). Results represent means of serum from a patient with complete C2 deficiency. three experiments. Intact bacteria, cell walls, and peptidoglycan were incubated for 60 min with 1% concentrations of normal, and that cell wall teichoic acid played little or no role in heat-inactivated, MgEGTA-chelated, C2-deficient, and this regard, electron microscope studies of thin sections heat-inactivated C2-deficient sera before adding PMN of cell walls and peptidoglycan (Fig. 1) had indicated leukocytes and measuring uptake at 15 min. Normal that these preparations differed morphologically as serum was the most effective opsonic source for intact well as in their previously defined chemical composi- bacteria, cell walls, and peptidoglycan, uptake being tions. Therefore, it seemed possible that removal of significantly greater than when either MNgEGTA- teichoic acid from the cell wall might have affected opsonization independently of the change brought *d S. aureus H about in chemical makeup. To investigate this possibil- 100r- O3 Ce// Wa//s ity, opsonization of a teichoic acid-deficient mutant ES Pepildog/ycan (52A5) was compared to that of its parent strain (HSmR) U)In 80K (Fig. 5). As shown, phagocytosis of the teichoic -

cL 60K A. Normol Serum B. Heat-inactivated Serum 100 0-) Parent Strain 0 40 H Q. In 80 20 H U-) 60 0' def def def def (D Opsonic Sources stan) Tn25(ecocai-elchcintlmutant)lpreincubate O' 20 FIGURE 6 PMN leukocyte uptake of intact S. aureus H, isolated cell walls, and purified peptidoglyean preincubated for 60 min with normal serum (NS), MgEGTA-chelated serum (X), heat-inactivated serum (AS), C2-deficient serum (C2 def) 5.01.0 01 5.01.0 01 5.01.0 01 5.0100.1 and heat-inactivated C2-deficient serum (AC2 def) before Serum Concentration (% adding leukocytes and determining uptake at 15 min. Results represent means of five experiments. Significance of differ- FIGURE 5 PMN leukocyte uptake of S. aureus HSMR (parent ences in uptake comparing NS and X, P < 0.001 for intact strain) and 52A5 (teichoic acid-deficient mutant) preincubated bacteria, cell walls and peptidoglycan; comparing NS and C2 with varying concentrations of (A) normal serum and (B) def, P < 0.025, 0.001, 0.001 for intact bacteria, cell walls and heat-inactivated serum before adding leukocytes and deter- peptidoglycan respectively; comparing C2 def and AC2 def, mining uptake at 15 min. Results represent means of three P < 0.001 for intact bacteria, cell walls and peptidoglyean (t experiments. test for paired data).

604 Peterson, Wilkinson, Kim, Verhoef, Schmeling, Douglas, and Quie chelated or C2-deficient sera were used as opsonic TABLE II sources (Fig. 6). Although MgEGTA-chelated serum Complement Activation in Normal Seruim by Peptidoglycatn * was not significantly more opsonic than heat- inactivated serum, C2-deficient serum had a greater Peptidoglycan CH,w opsonic capacity than heat-inactivated C2-deficient nig serum. 5.0 <10t These results suggested that opsoniization could 2.5 <10 proceed via a heat-labile pathway other than the 1.0 <10 classical pathway when C2 was blocked but not when 0.5 13 Cl was blocked. When the period of opsonization was 0.1 25 limited to 15 min rather than 60 min, however, 0.01 33 C2-deficient serum was no more effective than heat- Control 31 inactivated C2-deficient serum as an opsonic source, opsonization with the other serum sources not being * 1-mi aliquots of normal serum were incubated with equal affected by the shorter opsonization period (data not volumes of purified peptidoglycan, in concenitrations from 5.0 to 0.01 mg, and of water (control) for 30 min at 37°C. After ceni- presented). This finding indicated that opsonization via trifuging, CH50 was measured in the supernatant serum. the second heat-labile pathway proceeded more slowly t <10 = not detectable. than when opsonization was carried out in the presence of the classical pathway. Effect of lysostaphin treatment of peptidoglycan on was measured in the supernatant serum, and val- opsonizationi. To determine whether relatively small, ues were compared with C3-C9 levels measured soluble fragments of peptidoglycan could be opsonized in simultaneous controls (normal and C2-de- by normal and heat-inactivated sera, peptidoglycan was ficient sera incubated with water for the same treated with lysostaphin and the fragments incubated time intervals). Consumption of C3-C9 in the with 10% normal serum, 10% heat-inactivated serum, peptidoglycan-treated samples was expressed as a and HBSS before adding PMN leukocytes and measur- percentage relative to the C3-C9 levels in the controls ing leukocyte-associated radioactivity at 3, 10, and 15 (Fig. 7). As shown, activation of comiiplemnent occurred min. Whereas there was uptake of intact peptidoglycan 1)oth in the presence (normal serum) and in the absence in the control mixtures similar to that observed in (C2-deficient serum) of an intact classical pathway; earlier experiments (Fig. 2), no leukocyte-associated however, the rate of activation in C2-deficient serum radioactivity was recorded when peptidoglycan frag- was significantly slower than in nornmal serum (Fig. 7). ments were studied. These results suggested that for effective opsonization a certain particle size of the peptidoglycan was required. 100 _

C activation by peptidoglycan ( 80 Normal Serum Effect of peptidoglycan concentration on C activa- innrmal and C2-Deficient Serum tion itn niormal serum. As results from the above c60 experiments indicated that the peptidoglycan of S. 0 aureus H was the major C-activating component of the cell wall, additional studies were performed in which CL consumption of C by peptidoglycan was measured. 0 When purified peptidoglycan was incubated for 30 min 0 with normal serum in concentrations of from 5.0 to 0.01 2 mg/ml serum, a clear dose-response relationship was established (Table II). When concentrations of 1.0-5.0 0 mg were used, CH50 activity was not detectable. Kinetics of C activationi in normal and C2-deficient Minutes sera. The ability of peptidoglycan to activate C in the FIGURE 7 Kinetics of C activation by puirified peptidogly-can presence and in the absence of C2 was studied by in normal and C2-deficient sera. 1-miiI aliquots of normial and incubating normal and C2-deficient sera with peptidog- C2-deficient sera were incubated at 37°C with equal volumes of water (controls) and peptidoglx-can (1.0 mg) for 1, 5, 15, 60, lycan, at a concentration of 1.0 mg/ml, at 37°C. Samples and 120 min. Consumption of C3-C9 in the peptidoglycan- were removed at 1-, 5-, 15-, 60-, and 120-min intervals treated serum samples is expressed as a percent of the and centrifuged at 2°C. The hemolytic titer of C3-C9 simultaneous conitrol samples.

Opsonization of Staph ylococcal Peptidoglycan 6605 Effect of preabsorptiotn of serum with peptidoglycanl The relative importance of the peptidoglyean and on subsequent C activation. The ability of peptido- teichoic acid coomponents of the S. aureus cell wall in glyean to activate the C system in normal serum was the process of opsonizationi has been a matter of found to be significantly impaired when serum was controversy. Evidence has previously been presented preabsorbed with peptidoglycan at 2°C. Although there supporting the role of both teichoic acid (7-10) and( was 92, 89, and 37% conisumptioni of C3-C9 in control peptidoglyean (11, 12) in this regard. These studies, serum after a 30-min incubationi with 5.0, 1.0, and 0(.1 however, have used indirect methodls to assess opsonli- mg peptidoglycani, respectively, there was <25% zation, either bN, immunn izinig aiiinials with cell wall C3-C9 conistuinption in serunm that had been preab- components and measuring the protective effect of'such sorbed with peptidoglycan. Likewise, preabsorption of immunizationi (8, 9) or by absorbing seruml with cell C2-deficient serum with peptidoglycani at 2°C had an wall componients and determininig the effect of such inhibitory effect on the subse(uent capacity of pep- absorption on serum opsonic activity (7, 11, 12). tidoglycan to activate the C system. After 30-min and In this study, PMN leukocyte uptake of intact S. 60-min incubations with 1.0 mg peptidoglycan, there aureus H and of highly purified cell walls-composed was 9 and 44% C3-C9 consumption in nonabsorbed of peptidoglvcan and teichoic acid-and of isolatedl C2-deficient serum compared with 0 and 27% activation peptidoglycan was measured quantitatively using in C2-deficient serum which had been preabsorbed [3H]glycine-labeled l)acteria, cell walls, and peptido- with peptidoglycan. The absorption procedure itself glycan. When normal human serum and heat-inactivatedl did not significantly influence C3-C9 levels (after serum were used as opsoniic sources, intact bacteria, absorption, C3-C9 levels were 422 and 540 U/ml in cell walls, and peptidloglvcan were takeni up by PMIN normal and C2-deficient sera compared with control leukocytes in a simlilar manner. All three particles were values of 437 and 500 U/ml, respectively). These data most efficiently opsoniized bN normal serum, and uptake suggested that peptidoglycan activation of the C sys- did not occur in the absence of seruim factors. Electron tem, via both the classical and alternative pathways, mieroscope studies revealed that opson-iized peptido- depended upon the presenice of an absorbable, glycan was readily ingested by PM1N leukocytes. Studies nioncoimiplement-related serum factor. with purified IgG indicated that IgG was the miajor- heat-stable serumni opsonic factor, a finding reporte(d by DISCUSSION other investigators (44-46) for different staphylococcal strains. The cell wall of most S. aureuis strains is composed of As isolated cell wNalls (peptidoglycan and teichoic three major constituents -peptidoglycan, teichoic acid) and purified peptidoglycan were phagoeytized in acid, and protein A. The peptidoglycan component is a a comparable fashioin, it appeared that the remtoval of linear polymer of repeating ,l-I -4-linked N-acetylgltu- teichoic aci(l fromii the cell wvall did not significantly cosamine and N-acetylmuramic acid residues. Attached affect opsonization, thereby implying that the process to the carboxyl group of each N-acetylmuramic acid is a of opsonization wvas primarily inediated by pepti doglv- tetrapeptide cross-linked by a pentaglycine bridge to a cani. Electroni microscope stuidies, however, had re- neighboring peptide Unlit. Approximately 50% of the vealed that the cell wall anid peptidoglvcan prepara- weight of the cell wall is contributed by peptidoglycan, tions were nlot morphologically identical; in thini and it is this component which provides the wall its sections the peptidoglycani was, in generall, imiore basic rigidity (40). Cell wall teichoic acids, comprising collapsed. Therefore, the possibility existed that this -40% of cell wall weight, are charged polymers of structural difference might inifluience opsoniizationi in a ribitol-phosphate linked to muramic acid residues of manner independently of the well-defined chemiiical the peptidoglycan (41). Protein A, contributing -5% to difference of these particles. To help clarify this cell wall weight, is a protein with unique properties possibility, opsonization of' a teichoic acid-deficienit which, in general, interfere with the process of multanit straini of S. auireuis H was compared with that of opsonization (42, 43). In this investigationi, trypsin the parent strain. The abseniee of teichoic acid ini the digestioii was used to remove protein A and other cell cell wall di(l not alter opsoniizationi by niormiial and( Nwall proteins. Also lipoteichoic acid, which may be heat-inactivated sera which aclded support to the another comlponent of' the staphylococcal cell sturface hypothesis that teichoic acid played anl insignificant (18), was extracted, and the two major constituenits of role in bacterial opsonizationi. the cell wall -peptidoglycani and teichoic acid-were The findinig that there was no PMNI leukocyte uptake stuidied. The staphylococcal cell surface may be viewed of lysostaphin-degraded peptidoglycan which had been as a mosaic of different dcomains where all of the major preincubated with normal and heat-iniactivated sera cell wall components, inicluding peptidoglycan ancd stuggested that a certain particle size is re(Itqired for teichoic acid, can be sufficienitly exposedl to interact effective opsonizationi. Shavegani and colleagues (11) with external factors. 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 recently Streptococcus group A, Staphylococcus epidermidis, demonstrated that the iihibition of leukocyte migration and Micrococcus lysodeikticus were capable of activat- by staphylococcal peptidoglycan is also dependent ing both the classical and the alternative C pathways in upon the size of the peptidoglycan fragmiient. humnan seruim. Pryjma and colleagues (4) have demon- Other investigators have demonstrated that the strated the same capacity for S. aureus peptidoglycan classical (46, 48) and the alternative (46, 49, 50) C usinig serumil from guinea pigs and mice. pathways are involved in staphylococcal opsonizationi. In anl attempt to determine whether S. aureus In anl attempt to delineate which heat-labile serum peptidoglycan was capable of directly activating the factors participated in the opsonization of S. aureus H classical and alternative C pathways or whether C and of the peptidoglycan comiiponent, phagocytosis of activation was initiated by antigen-antibody complex intact bacteria, cell walls, and peptidoglycan was formation, normal and C2-deficient sera were preab- measured after opsonization with normal, MgEGTA- sorbed with peptidoglycan at 20C before adding chelated, heat-inactivated, C2-deficient, and heat- peptidoglycan at 37°C and measuring C3-C9 consump- inactivated C2-deficient sera. Opsonization of all three tion. Preabsorption of both serum sources with pep- particles was optimal when normal serum was used as tidoglyean was found to limit the ability of peptidogly- an opsonlic source. In the absence of C2, opsonization can to subsequently activate the classical and alterna- was less effective, both the opsonic capacity aind the tive pathways of C. These preliminary findings suggest rate of opsonization being less with C2-deficient serumil that C activation by peptidoglycan depends upon the than with normal sertum; however, opsonization did presence of an absorbable, noncomplement-related proceed via a heat-labile system (the opsonic capacity of factor, presumnabl immunoglobulin. Wheat and co- C2-deficient serum being significantly, reduced by workers (54) have presented data to stupport this hypothe- heat-inactivation). On the other hand, heat-labile sis. Activation of the classical C pathway, in general, is opsonic activity vas al)olished in the absence of Cl known to be initiated by the combining of IgG or IgNM (MgEGTA-chelated serumiii). with antigen (55). There is a growing body of evidence These results suggest that opsonization of intact S. which suggests that, in certain systems, activation of the aureuis H anid of peptidoglycan is most effective in the alternative complement pathway may likewise depend presenice of an intact classical pathway. However, it uiponl antigen-antibody formation (46, 56, 57). An- appears that opsonization can proceed via another tibodies of the IgG class, to S. aureus peptidoglyean heat-labile pathway when C2 is blocked but not wheni have been showin to be present in normal human serum Cl is blocked. Other investigators (51, 52) have (58). However, there is a need for studies utilizing demonstrated that C1 can play a role in the activation of purified C componeints to evaluate the possibility that the alternative complemiient pathway (the "Cl-bypass" peptidoglycain may directly interact with Cl and/or mechanism). From the results in this study, it appears directly activate the alternative C pathway as described that the C l-bypass mav be operative in the opsonization for certain gram-negative bacterial cell wall compo- of S. atureus H peptidoglycani by C2-deficient serum, nieints (59). and that blockiing Cl thereby interferes with alternative Over the past decade it has been demonstrated that pathway activation. the recognition of staphylococci by PMN leukocytes is Activationi of the serumii C system by peptidoglvcan mediated by receptors within the phagocytic mem- was further studied bv incubating peptidoglycan with brane with specificity for the Fc fragment of IgG and for normal serumiii anid miieasuring C constumption. A (lose- anl activated form of the third component of C, C3b (60, response relationiship was established in which CH5, 61). Staphylococcal peptidoglycan would appear to play was not detectable after a 30-mmliil incubation with a ceentral role in the initiation of this recognition peptidoglvycan in conicentrationis of 1.0-5.0 mg/ml seninm. process. Other investigators (1, 62) have proposed that The kinetics of C activation were then investigatedl the peptidoglycan, a maijor cell wall component of most usinlg normlal an(l C2-deficieint sera. Although the rate of granm-positive bacteria, may play a significant role in the C3-C9 coinstumiiptioni Vwcas significanfly faster and pro- developmenit of' "natural immunity" to this grouip of ceede(d to a greater degree in niormiial serumii, C3-C9 was miieroorganisms. Recognition of all gram-positive bac- also consumiied in C2-deficient serumii. These findings teria may, therefore, be a manifestation of host response were consistenit with observations miade in the funic- to this comminioni cell wall conmponent. tionIal phagocytosis assay whereinl C2-deficient seruiml It shouild be emphasized that although cell wall was nlot as effective as normiial serum as an Opsfoiic teichoic acicl was found not to be important in source aiid opsonlizatioin proceeded at a slower rate in promlotinig opsonization in the serunm sources used in C2-deficient serumni than in niornmal serumiii. Simiiilar this stuLdyl, the possibility remains that teichoic acid is of findinigs have beeni reported in studies of opsoniizationi imlportance in fostering opsonization in serunm froIml of other staphylococcal strainis (28, 42). Recentl-, patienits with S. auireus iinfections in which inereased Bokisch (53) has reported that peptidoglycans froml levels of teichoic acid anitibodies have been demnon-

Opson ization of Staphyllococcal Peptidoglycan 6076 strate(d (63). Futuire stu(lies vill be directed at inves- resistanit imutitanit of Staphi lococcus alertus. 1. Bacteriol. tigatinig this possibility. 11 1: 272-283. 1.5. Shaw, D. R. D., D. Nlirelniaii, A. N. Chatterjec, and J.l T. Park. 1970. Ribitol teichoic acidl synthesis ill bac- ACKNOWLE l)G ME NTS teriophage-resistanit muttanits of'Staphillococciis atirenis H. 'I'l)c auitlhors aire gratefull to D. Petersoni f'or the aIIiii0 acid 1. Biol. Chern. 245: 5101-5106. analyses, to Patti Lorenz f'or her secretarial assistancie, anid to L. 16. Gilpini, R. \V., A. N. Chatterjee, and( F. E. Young. 1972. D. Sabath for hiis valuable discuissioni aind suipport. Auitolvsis of' microbial cells: salt activation of autol\ tic This work wasstupportecl hv U. S. Public Health Service enzymes in a mutitanlt of Staphylococcs.s (Itl)CIs. 1. graniits Al 06931-11, Al Al Bacteriol. 111: 272-283. AI-08821-07, 12478-03, 10704, anid 17. 1973. 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