INFECTION AND IMMUNITY, May 1978, p. 388-392 0019-9567/78/0020-0388$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 20, No. 2
Printed in U.S.A.
Activation of Complement by Cell Surface Components of Staphylococcus aureus BRIAN J. WILKINSON,2* YOUNGKI KIM,3 PHILLIP K. PETERSON,' PAUL G. QUIE,2" AND ALFRED F. MICHAEL" Departments of Medicine,' Microbiology,2 and Pediatrics,3 University of Minnesota, Medical School, Mayo Memorial Building, Minneapolis, Minnesota 55455 Received for publication 16 January 1978
The abilities of intact Staphylococcus aureus H, crude cell walls (CCW), purified cell walls (PCW, peptidoglycan [PG] and covalently linked teichoic acid), peptidoglycan, and cell membranes (CM) to activate the complement system in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum were compared. On a weight basis, PCW was the most active fraction; intact organisms and CCW were about equally effective; and PG was least active in causing complement consumption in normal serum. CM also activated complement but did not give a clear dose-response relationship in the concentrations used. Kinetic studies revealed that C3-C9 consumption occurred at a significantly slower rate in C2-deficient serum, indicating that intact organisms, PCW, and PG may activate the complement system via the classical and alternative pathways in normal serum. C3-C9 consumption was also slower in immunoglobulin-deficient serum than in normal serum, implying that immunoglobulins play a role in attaining maximum rates of complement activation. In all sera studied, PG was less active in complement activation than PCW. These results indicate that a number of cell surface components of S. aureus can play a role in complement activation by this organism and that the presence of teichoic acid has a significant enhancing effect in this regard. Recent studies in this laboratory have focused attention on peptidoglycan (PG) as being the Staphylococcus aureus cell surface component of key importance in promoting opsonization of this organism by both heat-labile and heat-stable factors in nonimmune human serum (P. K. Peterson, B. J. Wilkinson, Y. Kim, J. Verhoef, D. Schmeling, S. D. Douglas, and P. G. Quie, J. Clin. Invest., in press). Isolated PG was shown to activate both the classical and alternative complement pathways, a finding consistent with the results of other workers (2, 11). In addition to PG, the cell surface of most S. aureus strains contains teichoic acid (TA) and protein A, the other two major cell wall polymers (1). PG is a cross-linked heteropolymer of amino acids and amino sugars that makes up about 50% of cell wall weight. TA, which contributes about 40% of cell wall weight, is a negatively charged ribitol-phosphate polymer with N-acetyl-D-glucosamine and D-alanine substituents that is covalently attached to PG through a short chain of glycerol phosphate residues and probably an N-acetyl-D-glucosamine residue (3). Protein A is also covalently linked to PG (12) and makes up about 5% of the cell wall weight. Besides the cell wall, elements of the cell mem-
brane, including lipoteichoic acid (15), may form part of the staphylococcal cell surface. Recently, other investigators have examined complement activation by streptococcal cell surface components (14, 17, 18). The purpose of the present investigation was to determine the role of several staphylococcal cell surface components in activation of the serum complement system. Complement consumption by intact bacteria was compared with that elicited by crude cell walls (CCW), prepared in a manner designed to leave the cell surface relatively undegraded, purified cell walls (PCW), composed of PG with covalently linked TA, isolated PG, and cell membrane fragments (CM). Complement activation was studied in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum. Although all cell surface fractions studied were capable of activating complement, dose-response and kinetic studies revealed PCW to be the most active fraction.
MATERIALS AND METHODS Organism and cultural conditions. S. aureus H
was used because of its well-established cell wall structure (1). Bacteria were grown with aeration in peptoneyeast extract broth (6), and late exponential-phase organisms were used.
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Preparation of cell surface fractions. PCW were prepared by sodium dodecyl sulfate, nuclease, trypsin, and phenol treatments, and PG was isolated from PCW by hot trichloroacetic acid extraction, as previously described (P. K. Peterson et al., J. Clin. Invest., in press). CCW were prepared by breaking organisms with glass beads in a Vibrogen cell mill (R.H.O. Scientific Inc., Commack, N.J.) and isolating the walls by differential centrifugation. The walls were washed six times in distilled water and lyophilized. From about 4 g (dry weight) of organisms, 950 mg of CCW was recovered. To isolate CM, about 4 g (dry weight) of organisms was washed once in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.5, at 22°C, resuspended in 200 ml of 0.05 M Tris-hydrochloride, pH 7.5, containing a solution of 0.145 M NaCl, 0.05 M Mg9l2, 1.0 M sucrose, and lysostaphin (25 ,ug/ml, 210 U/mg; Schwarz/Mann, Orangeburg, N.Y.), and incubated at 37°C for 1 h. The suspension was then centrifuged, resuspended in 200 ml of 0.05 M Tris-hydrochloride containing 5 ig of deoxyribonuclease I per ml (EC 3.1.4.5.; Worthington Biochemical Corp., Freehold, N. J.), and stirred at 22°C for 15 min to lyse the organisms. CM were deposited by centrifuging (39,000 x g for 30 min at 2°C), washed five times in distilled water, and lyophilized. The yield of CM was 530 mg. Intact organisms were freshly grown and washed once in cold distilled water before being used in studies of complement activation. Chemical characterization of cell surface fractions. The methods for this have been detailed previously (P. K. Peterson et al., J. Clin. Invest., in press). Amino acid and amino sugar contents of preparations were estimated on an amino acid analyser after hydrolysis in 6 M HCl for 18 h at 105°C. Phosphorus was estimated colorimetrically and fatty acids by gas-liquid chromatography after saponification and methylation. Protein A estimation. The protein A content of S. aureus H was measured by using an indirect hemagglutination assay with sensitized sheep erythrocytes (16) as previously described (10). Measurement of C3-C9 consumption. Fifty-microliter aliquots of distilled water (control) and 50-jd aliquots of appropriate concentrations of intact organisms and the various fractions were added to 0.5-ml aliquots of serum. The mixtures were incubated in a shaking incubator (New Brunswick Scientific, New Brunswick, N.J.) at 37°C and 250 rpm for indicated times before centrifuging (10,000 x g for 30 min at 2°C). Mixtures containing CM were centrifuged at 16,000 x g for 30 min at 2°C. The supemnatants were stored at -70°C until C3-C9 hemolytic assays were performed by methods previously described (5, 9). Test samples were serially diluted in 0.01 M ethylenediaminetetraacetic acid (EDTA)-gelatin-glucose-Veronal buffer without magnesium and calcium (EDTAGGVB--) from 1:20 to 1:640. One milliliter of 0.01 M EDTA-GGVB-- was added to 1.0 ml of each dilution of test samples. Hemolytic cellular intermediates (2a) were prepared at Tmax using Cl (guinea pig), C4 (human), and C2 (guinea pig), and 1.0 ml of EAC142 (5 x 107/ml) was then added to each tube. After incubation at 37°C for 60 min, 4.5 ml of cold 0.9% (wt/vol) saline was added and the tubes were centrifuged. The
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optical density of the supematants was read at 412 nm, and the results are expressed as 50% lysis. Appropriate controls and complete blanks were run simultaneously. Serum sources. Normal human sera were obtained from five healthy donors, who denied previous staphylococcal infection, and were pooled. Serum was obtained from a donor with a genetically determined complete and selective deficiency of C2 (7). Immunoglobulin-deficient serum was obtained from a 62-yearold male with common variable immunodeficiency (immunoglobulin G, 73 mg; -M, 7 mg; and -A, 3 mg/100 ml by laser nephelometry). This serum had normal levels of all complement components except for a somewhat diminished hemolytic titer of Cl (96,000 hemolytic U/ml; 126,000 to 369,000 hemolytic U/ml, normal range). Sera were stored in fractions at -70°C before use.
RESULTS Chemistry of cell surface fractions. The chemistry of the PCW and PG used in this study has been described previously (P. K. Peterson et al., J. Clin. Invest., in press). PG plus TA components accounted for 91.5% of the PCW, and 92.4% of the PG weight was accounted for as PG components. PG contained 5% of the cell wall phosphate, indicating this preparation was free of TA. Fatty acids were present in PCW and PG preparations at levels less than 1 nmol/mg, indicating very low contamination by membranes and/or lipoteichoic acid. Electron microscopic examination of PCW and PG (P. K. Peterson et al., J. Clin. Invest., in press) indicated excessive comminution of the particles had not occurred during cell breakage. In other words, one intact organism generally yielded one cell wall particle. Morphologically, PG was somewhat more collapsed than PCW. The analyses of the CCW and CM are given in Table 1. CCW are composed primarily of PG and TA. Large amounts of protein did not appear to be present as indicated by low levels of non-PG amino acids. The full range of protein amino acids was found in analyses of CM and was present at levels that indicate that CM are 60 to 70% protein by weight. The presence of fatty acids in appreciable amounts in CM is consistent with the expected high lipid content of this fraction. The intact bacteria contained protein A as revealed by a titer of 1:640 by indirect hemagglutination. S. aureus Cowan I and S. aureus Wood 46, a protein A-deficient strain, had titers of 1:1,280 and 0, respectively. CCW were expected to contain protein A as proteolytic digestion was not used in their preparation. Under the analytical conditions used, a full complement of protein amino acids was not detected. This may be due to the low protein A content of S. aureus walls by weight (12) that therefore yield
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INFECT. IMMUN.
TABLE 1. Chemistry of S. aureus H CCW and CM" Component
Cysteic acid Aspartic acid
............ ...........
Threonime Serine Muramic acid ........... Glutamic acid ........... Proline ................. Glycine ................ Alanine ................ Valine ................. Methionine ............. Isoleucine .............. Leucine ................ Glucosamine ............ Tyrosine ............... Phenylalanine .......... Lysine ................. Histidine ...............
Arginine ................ Phosphate ..............
CCW CM (nmol/mg (nmol/mg [dry weight]) [dry weight])
8 44 24 48 236 512 6
2,320 1,932 24
Tr 20 28 752
NDb ND 512 ND 12 880 40.4
36 588 312 272 Tr 644 176 604 648 360 8 352 436 68 116 208 440 120 208 550 190
way. However, whereas significant C3-C9 consumption occurred in normal serum within 5 min of incubation, it was not observed until after 15 min in C2-deficient serum. C3-C9 consumption in immunoglobulin-deficient serum by all three preparations was slower than in normal serum, but faster than in C2-deficient serum. In normal human serum, PCW caused more rapid complement consumption than intact organisms; in the other two sera these preparations were about equally active. However, in all three sera, PG was clearly less active than the other two preparations.
DISCUSSION In this study, all S. aureus H cell surface fractions tested were found capable of activating serum complement. In normal human serum, PCW caused more complement consumption than an equal weight of intact organisms (Fig. 1 and 2a). However, since the cell wall comprises
Fatty acid .............. a The cell fractions were prepared and analyzed as described in Materials and Methods. b ND, Not detected.
low amounts of protein A amino acids on hydrolysis. Proteolytic digestion is known to remove protein A (12), and this was used during preparation of PCW. C3-C9 activation in normal human serum by intact bacteria and cell surface fractions. Intact bacteria and cell surface fractions, in weights from 0.05 to 2.5 mg, were added to 0.5 ml of normal human serum and incubated for 1 h at 370C. C3-C9 consumption in the treated samples was expressed as a percentage relative to the C3-C9 levels in the control. All of the preparations led to some complement consumption in 1 h at concentrations as low as 10 ,ug of serum per ml. The results show that, on a weight basis, PCW were the most active fractions in causing C3-C9 consumption; CCW and intact organisms were about equally active; and PG was least active. Since PCW were considerably more active in complement consumption than PG, this indicates that the presence of TA significantly enhances complement activation. Kinetics of C3-C9 consumption by intact bacteria, PCW, and PG in normal, C2-deficient, and immunoglobulin-deficient sera. The kinetics of C3-C9 consumption by intact bacteria, PCW, and PG in various sera were determined (see Fig. 2). In C2-deficient serum intact organisms, PCW, and PG activated complement, indicating that all three preparations can activate the alternative complement path-
100
Ce/I
re)
.
nl 01
-0.01
0.1
J10
1.0
Component Concentration (mg *y wi /mi serum )
FIG. 1. C3-C9 consumption by S. aureus Hand cell surface fractions in normal human serum. Various weights of intact organisms and fractions were incubated with serum for 60 min at 37°C before determination of C3-C9 consumption.
S'S (a) Normel Serum
(b) C2-eficient Serum
(D)" m Sbuleun-
E 00
c M
05
10 15
Mo51
Mo
105
M
Time (min)
FIG. 2. Kinetics of C3-C9 consumption by S. aureus H PCW and PG in various serum sources. Intact organisms, PCW, and PG were incubated in normal serum (a), C2-deficient serum (b), and immunoglobu-
lin-deficient serum (c) for specified times at a concentration of 1 mg of dry weight/ml of serum. C3-C9 consumption was determined as described in Materials and Methods with a parallel distilled water control included at each time point.
VOL. 20, 1978
STAPHYLOCOCCAL SURFACE AND COMPLEMENT
about 20% of the dry weight of the cell, there would be about five times as many particles or "cell wall" in a PCW preparation as in an equal weight of intact organisms. Thus, if the results were evaluated on an efficiency per particle basis, the intact organisms and PCW would not appear to be greatly different in activity. Consumption of C3-C9 by CCW was less than that by PCW, suggesting that minor cell surface components did not augment C3-C9 consumption. Isolated protein A is capable of activating the alternative and classical pathways of complement (13), and we would perhaps have anticipated CCW to retain this component and be more active than PCW. It is possible that when protein A is in the cell wall it is not as active as when in the isolated state. Also, complement consumption by isolated protein A has been shown to occur in a paradoxical manner where increasing the protein A concentrations beyond an optimal level results in reduced complement consumption-(8, 13). On a weight basis, CM were not as active in complement consumption as intact bacteria. They would seem to be even less active if compared on the basis of the number of organisms required to yield a given weight of CM, since the cell membrane comprises about 10 to 15% of the weight of the staphylococcus. It is unclear to what extent the cell membrane contributes to complement activation by intact organisms, in that the membrane is expected to be somewhat shielded by the cell wall. When complement consumption by PG in normal serum was studied this fraction was clearly not as active as either intact organisms or PCW when equal weights of the preparations were used. The PG was even less active on a particle basis since PG comprises about 50% of cell wall weight and there would, therefore, be about 10 PG particles for every intact organism. In addition, when kinetic studies were performed, complement was activated at a significantly slower rate by PG than by PCW or intact organisms. The essential difference between PCW and PG is the presence of TA and the linkage unit in the former preparations. Thus it appears that TA covalently attached to PG markedly enhances complement activation. In a previous study (P. K. Peterson, et al., J. Clin. Invest., in press) we were unable to demonstrate a significant role for TA in staphylococcal opsonization by the complement system, and PG was the key cell surface component in this regard. Thus, it would appear that the phenomena of bacterial opsonization and complement consumption cannot be automatically equated. Similarly, we have recently found an encapsulated
391
S. aureus strain to significantly activate complement and yet be ineffectively opsonized (lOa). In a kinetic study of chemotactic factor generation in human serum by complement activation, Gallin et al. (4) showed that chemotactic factor generation proceeded more rapidly when mediated by the classical pathway than by the alternative pathway. Those authors concluded that activation by the classical and alternative pathways could be distinguished by kinetic analysis. Since C3-C9 consumption by intact bacteria, PCW, and PG in normal serum was more rapid than in C2-deficient serum, this indicates that both the classical and alternative pathways may be involved in complement activation in normal serum. In serum relatively deficient in antibodies, C3-C9 consumption by the preparations occurred at a significantly slower rate than in normal serum, but somewhat faster than in C2deficient serum. These results suggest that immunoglobulins are necessary to obtain maximal rates of complement activation. Further studies will be needed to delineate the role immunoglobulins play in activation of the classical and/or alternative pathways. Winkelstein and Tomasz (17, 18) evaluated the role of Streptococcus pneumoniae cell surface structures in activating the alternative pathway in C4-deficient guinea pig serum. These authors found that their CCW preparations were less active than PCW and that TA appeared to mediate complement consumption in this serum. S. pneumoniae PG was reported to be inactive in complement consumption. Our results using C2-deficient serum support a significant role for TA in activation of the alternative pathway. However, we found, as have others (2, 11), that staphylococcal PG can activate the alternative pathway, albeit at a slow rate. From these studies it is clear that several S. aureus cell surface components may participate in activation of serum complement. Also, both the classical and alternative pathways can be activated by these components. Further elucidation of the interaction of the staphylococcal cell surface and the serum complement system would appear to be of fundamental importance in understanding host reaction to staphylococcal infection. ACKNOWLEDGMENTS This work was supported by Public Health Service grants AI 6931-11, Al 08821-07, and Al 10704, from the National Institute of Allergy and Infectious Diseases, HL 06314, from the National Heart, Lung and Blood Institute, and by two Grants-in-Aid from the University of Minnesota Graduate School. P. G. Quie is the American Legion Memorial Heart Research Professor. We are grateful to Shirley Hermel for her secretarial help.
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WILKINSON ET AL. LITERATURE CITED
1. Archibald, A. R. 1972. The chemistry of staphylococcal cell walls, p. 75-109. In J. 0. Cohen (ed.), The staphylococci. Wiley Interscience, New York. 2. Bokish, V. 1975. Interaction of peptidoglycans with antiIgGs and with complement. Z. Immunitaetsforsch. Exp. Klin. Immunol. 149:320-330. 2a. Boros, T., and H. J. Rapp. 1967. Immune hemolysis: a simplified method for the preparation of EAC'4 with guinea pig or with human complement. J. Immunol. 99:263-268. 3. Coley, J., A. R. Archibald, and J. Baddiley. 1977. The presence of N-acetylglucosamine 1-phosphate in the linkage unit that connects teichoic acid to peptidoglycan in Staphylococcus aureus. FEBS Lett. 80:405-407. 4. Gallin, J. I., R. A. Clark, and M. M. Frank. 1975. Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways. Clin. Immunol. Immunopathol. 3:334-346. 5. Gewurz, H., A. R. Page, R. J. Pickering, and R. Good. 1967. Complement activity with glomerulonephritis, essential hypocomplementemia and agammaglobulinemia. Int. Arch. Allergy Appl. Immunol. 32:64-90. 6. Gilpin, R. W., A. N. Chatterjee, and F. E. Young. 1972. Autolysis of microbial cells, salt activation of autolytic enzymes in a mutant of Staphylococcus aureus. J. Bacteriol. 111:272-283. 7. Kim, Y., P. S. Friend, I. G. Dresner, E. J. Yunis, and A. F. Michael. 1977. Inherited deficiency of the second component of complement (C2) with membranoproliferative glomerulonephritis. Am. J. Med. 62:765-771. 8. Kronvall, G., and H. Gewurz. 1970. Activation and inhibition of IgG mediated complement fixation by staphylococcal protein A. Clin. Exp. Immunol. 7:211-220. 9. McLean, R. G., H. Geiger, B. Burke, R. Simmons, J. Najarian, R. L. Vernier, and A. F. Michael. 1976. Recurrence of membranoproliferative glomerulonephri-
INFECT. IMMUN. tis following kidney transplantation Am. J. Med. 60:60-72. 10. Peterson, P. K., J. Verhoef, L. D. Sabath, and P. G. Quie. 1977. Effect of protein A on staphylococcal opsonization. Infect. Immun. 15:760-764. lOa. Peterson, P. K., B. J. Wilkinson, Y. Kim, D. Schmeling, and P. G. Quie. 1978. Influence of encapsulation on staphylococcal opsonization and phagocytosis by human polymorphonuclear leukocytes. Infect. Immun. 19:943-949. 11. Pryjma, J., K. Pryjma, A. Grov, and P. B. Heczko. 1976. Immunological activity of staphylococcal cell wall antigens, p. 873-881. In J. Jeljasweicz, (ed.), Staphylococci and staphylococcal diseases. Gustav Fischer Verlag, New York. 12. Sjoquist, J. 1973. Structure and immunology of protein A, p. 83-92. In J. Jelaszewicz, (ed.), Contributions to microbiology and immunology, vol. 1: staphylococci and staphylococcal infections. Karger, Basel. 13. Stilenheim, G., 0. Goxtze, N. R. Cooper, J. Sjoquist, and H. J. Muller-Eberhard. 1973. Consumption of human complement components by complexes of IgG with protein A of Staphylococcus aureus. Immunochemistry 10:501-507. 14. Tauber, J. W., M. J. Poiley, and J. B. Zabriskie. 1976. Nonspecific complement activation by streptococcal structures. II. Properidin-independent initiation of the alternative pathway. J. Exp. Med. 143:1352-1366. 15. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acids: a new class of bacterial antigens. Science 187:1161-1167. 16. Winblad, S., and C. Ericson. 1973. Sensitized sheep red cells as a reactant for Staphylococcus aureus protein A. Acta Pathol. Microbiol. Scand. Sect. B 81:150-156. 17. Winkelstein, J. A., and A. Tomasz. 1977. Activation of the alternative pathway by pneumococcal cell walls. J. Immunol. 118:451-454. 18. Winkelstein, J. A., and A. Tomasz. 1978. Activation of the alternative complement pathway by pneumococcal cell wall teichoic acid. J. Immunol. 120:174-178.
Vol. 20, No. 2
Printed in U.S.A.
Activation of Complement by Cell Surface Components of Staphylococcus aureus BRIAN J. WILKINSON,2* YOUNGKI KIM,3 PHILLIP K. PETERSON,' PAUL G. QUIE,2" AND ALFRED F. MICHAEL" Departments of Medicine,' Microbiology,2 and Pediatrics,3 University of Minnesota, Medical School, Mayo Memorial Building, Minneapolis, Minnesota 55455 Received for publication 16 January 1978
The abilities of intact Staphylococcus aureus H, crude cell walls (CCW), purified cell walls (PCW, peptidoglycan [PG] and covalently linked teichoic acid), peptidoglycan, and cell membranes (CM) to activate the complement system in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum were compared. On a weight basis, PCW was the most active fraction; intact organisms and CCW were about equally effective; and PG was least active in causing complement consumption in normal serum. CM also activated complement but did not give a clear dose-response relationship in the concentrations used. Kinetic studies revealed that C3-C9 consumption occurred at a significantly slower rate in C2-deficient serum, indicating that intact organisms, PCW, and PG may activate the complement system via the classical and alternative pathways in normal serum. C3-C9 consumption was also slower in immunoglobulin-deficient serum than in normal serum, implying that immunoglobulins play a role in attaining maximum rates of complement activation. In all sera studied, PG was less active in complement activation than PCW. These results indicate that a number of cell surface components of S. aureus can play a role in complement activation by this organism and that the presence of teichoic acid has a significant enhancing effect in this regard. Recent studies in this laboratory have focused attention on peptidoglycan (PG) as being the Staphylococcus aureus cell surface component of key importance in promoting opsonization of this organism by both heat-labile and heat-stable factors in nonimmune human serum (P. K. Peterson, B. J. Wilkinson, Y. Kim, J. Verhoef, D. Schmeling, S. D. Douglas, and P. G. Quie, J. Clin. Invest., in press). Isolated PG was shown to activate both the classical and alternative complement pathways, a finding consistent with the results of other workers (2, 11). In addition to PG, the cell surface of most S. aureus strains contains teichoic acid (TA) and protein A, the other two major cell wall polymers (1). PG is a cross-linked heteropolymer of amino acids and amino sugars that makes up about 50% of cell wall weight. TA, which contributes about 40% of cell wall weight, is a negatively charged ribitol-phosphate polymer with N-acetyl-D-glucosamine and D-alanine substituents that is covalently attached to PG through a short chain of glycerol phosphate residues and probably an N-acetyl-D-glucosamine residue (3). Protein A is also covalently linked to PG (12) and makes up about 5% of the cell wall weight. Besides the cell wall, elements of the cell mem-
brane, including lipoteichoic acid (15), may form part of the staphylococcal cell surface. Recently, other investigators have examined complement activation by streptococcal cell surface components (14, 17, 18). The purpose of the present investigation was to determine the role of several staphylococcal cell surface components in activation of the serum complement system. Complement consumption by intact bacteria was compared with that elicited by crude cell walls (CCW), prepared in a manner designed to leave the cell surface relatively undegraded, purified cell walls (PCW), composed of PG with covalently linked TA, isolated PG, and cell membrane fragments (CM). Complement activation was studied in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum. Although all cell surface fractions studied were capable of activating complement, dose-response and kinetic studies revealed PCW to be the most active fraction.
MATERIALS AND METHODS Organism and cultural conditions. S. aureus H
was used because of its well-established cell wall structure (1). Bacteria were grown with aeration in peptoneyeast extract broth (6), and late exponential-phase organisms were used.
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Preparation of cell surface fractions. PCW were prepared by sodium dodecyl sulfate, nuclease, trypsin, and phenol treatments, and PG was isolated from PCW by hot trichloroacetic acid extraction, as previously described (P. K. Peterson et al., J. Clin. Invest., in press). CCW were prepared by breaking organisms with glass beads in a Vibrogen cell mill (R.H.O. Scientific Inc., Commack, N.J.) and isolating the walls by differential centrifugation. The walls were washed six times in distilled water and lyophilized. From about 4 g (dry weight) of organisms, 950 mg of CCW was recovered. To isolate CM, about 4 g (dry weight) of organisms was washed once in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.5, at 22°C, resuspended in 200 ml of 0.05 M Tris-hydrochloride, pH 7.5, containing a solution of 0.145 M NaCl, 0.05 M Mg9l2, 1.0 M sucrose, and lysostaphin (25 ,ug/ml, 210 U/mg; Schwarz/Mann, Orangeburg, N.Y.), and incubated at 37°C for 1 h. The suspension was then centrifuged, resuspended in 200 ml of 0.05 M Tris-hydrochloride containing 5 ig of deoxyribonuclease I per ml (EC 3.1.4.5.; Worthington Biochemical Corp., Freehold, N. J.), and stirred at 22°C for 15 min to lyse the organisms. CM were deposited by centrifuging (39,000 x g for 30 min at 2°C), washed five times in distilled water, and lyophilized. The yield of CM was 530 mg. Intact organisms were freshly grown and washed once in cold distilled water before being used in studies of complement activation. Chemical characterization of cell surface fractions. The methods for this have been detailed previously (P. K. Peterson et al., J. Clin. Invest., in press). Amino acid and amino sugar contents of preparations were estimated on an amino acid analyser after hydrolysis in 6 M HCl for 18 h at 105°C. Phosphorus was estimated colorimetrically and fatty acids by gas-liquid chromatography after saponification and methylation. Protein A estimation. The protein A content of S. aureus H was measured by using an indirect hemagglutination assay with sensitized sheep erythrocytes (16) as previously described (10). Measurement of C3-C9 consumption. Fifty-microliter aliquots of distilled water (control) and 50-jd aliquots of appropriate concentrations of intact organisms and the various fractions were added to 0.5-ml aliquots of serum. The mixtures were incubated in a shaking incubator (New Brunswick Scientific, New Brunswick, N.J.) at 37°C and 250 rpm for indicated times before centrifuging (10,000 x g for 30 min at 2°C). Mixtures containing CM were centrifuged at 16,000 x g for 30 min at 2°C. The supemnatants were stored at -70°C until C3-C9 hemolytic assays were performed by methods previously described (5, 9). Test samples were serially diluted in 0.01 M ethylenediaminetetraacetic acid (EDTA)-gelatin-glucose-Veronal buffer without magnesium and calcium (EDTAGGVB--) from 1:20 to 1:640. One milliliter of 0.01 M EDTA-GGVB-- was added to 1.0 ml of each dilution of test samples. Hemolytic cellular intermediates (2a) were prepared at Tmax using Cl (guinea pig), C4 (human), and C2 (guinea pig), and 1.0 ml of EAC142 (5 x 107/ml) was then added to each tube. After incubation at 37°C for 60 min, 4.5 ml of cold 0.9% (wt/vol) saline was added and the tubes were centrifuged. The
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optical density of the supematants was read at 412 nm, and the results are expressed as 50% lysis. Appropriate controls and complete blanks were run simultaneously. Serum sources. Normal human sera were obtained from five healthy donors, who denied previous staphylococcal infection, and were pooled. Serum was obtained from a donor with a genetically determined complete and selective deficiency of C2 (7). Immunoglobulin-deficient serum was obtained from a 62-yearold male with common variable immunodeficiency (immunoglobulin G, 73 mg; -M, 7 mg; and -A, 3 mg/100 ml by laser nephelometry). This serum had normal levels of all complement components except for a somewhat diminished hemolytic titer of Cl (96,000 hemolytic U/ml; 126,000 to 369,000 hemolytic U/ml, normal range). Sera were stored in fractions at -70°C before use.
RESULTS Chemistry of cell surface fractions. The chemistry of the PCW and PG used in this study has been described previously (P. K. Peterson et al., J. Clin. Invest., in press). PG plus TA components accounted for 91.5% of the PCW, and 92.4% of the PG weight was accounted for as PG components. PG contained 5% of the cell wall phosphate, indicating this preparation was free of TA. Fatty acids were present in PCW and PG preparations at levels less than 1 nmol/mg, indicating very low contamination by membranes and/or lipoteichoic acid. Electron microscopic examination of PCW and PG (P. K. Peterson et al., J. Clin. Invest., in press) indicated excessive comminution of the particles had not occurred during cell breakage. In other words, one intact organism generally yielded one cell wall particle. Morphologically, PG was somewhat more collapsed than PCW. The analyses of the CCW and CM are given in Table 1. CCW are composed primarily of PG and TA. Large amounts of protein did not appear to be present as indicated by low levels of non-PG amino acids. The full range of protein amino acids was found in analyses of CM and was present at levels that indicate that CM are 60 to 70% protein by weight. The presence of fatty acids in appreciable amounts in CM is consistent with the expected high lipid content of this fraction. The intact bacteria contained protein A as revealed by a titer of 1:640 by indirect hemagglutination. S. aureus Cowan I and S. aureus Wood 46, a protein A-deficient strain, had titers of 1:1,280 and 0, respectively. CCW were expected to contain protein A as proteolytic digestion was not used in their preparation. Under the analytical conditions used, a full complement of protein amino acids was not detected. This may be due to the low protein A content of S. aureus walls by weight (12) that therefore yield
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INFECT. IMMUN.
TABLE 1. Chemistry of S. aureus H CCW and CM" Component
Cysteic acid Aspartic acid
............ ...........
Threonime Serine Muramic acid ........... Glutamic acid ........... Proline ................. Glycine ................ Alanine ................ Valine ................. Methionine ............. Isoleucine .............. Leucine ................ Glucosamine ............ Tyrosine ............... Phenylalanine .......... Lysine ................. Histidine ...............
Arginine ................ Phosphate ..............
CCW CM (nmol/mg (nmol/mg [dry weight]) [dry weight])
8 44 24 48 236 512 6
2,320 1,932 24
Tr 20 28 752
NDb ND 512 ND 12 880 40.4
36 588 312 272 Tr 644 176 604 648 360 8 352 436 68 116 208 440 120 208 550 190
way. However, whereas significant C3-C9 consumption occurred in normal serum within 5 min of incubation, it was not observed until after 15 min in C2-deficient serum. C3-C9 consumption in immunoglobulin-deficient serum by all three preparations was slower than in normal serum, but faster than in C2-deficient serum. In normal human serum, PCW caused more rapid complement consumption than intact organisms; in the other two sera these preparations were about equally active. However, in all three sera, PG was clearly less active than the other two preparations.
DISCUSSION In this study, all S. aureus H cell surface fractions tested were found capable of activating serum complement. In normal human serum, PCW caused more complement consumption than an equal weight of intact organisms (Fig. 1 and 2a). However, since the cell wall comprises
Fatty acid .............. a The cell fractions were prepared and analyzed as described in Materials and Methods. b ND, Not detected.
low amounts of protein A amino acids on hydrolysis. Proteolytic digestion is known to remove protein A (12), and this was used during preparation of PCW. C3-C9 activation in normal human serum by intact bacteria and cell surface fractions. Intact bacteria and cell surface fractions, in weights from 0.05 to 2.5 mg, were added to 0.5 ml of normal human serum and incubated for 1 h at 370C. C3-C9 consumption in the treated samples was expressed as a percentage relative to the C3-C9 levels in the control. All of the preparations led to some complement consumption in 1 h at concentrations as low as 10 ,ug of serum per ml. The results show that, on a weight basis, PCW were the most active fractions in causing C3-C9 consumption; CCW and intact organisms were about equally active; and PG was least active. Since PCW were considerably more active in complement consumption than PG, this indicates that the presence of TA significantly enhances complement activation. Kinetics of C3-C9 consumption by intact bacteria, PCW, and PG in normal, C2-deficient, and immunoglobulin-deficient sera. The kinetics of C3-C9 consumption by intact bacteria, PCW, and PG in various sera were determined (see Fig. 2). In C2-deficient serum intact organisms, PCW, and PG activated complement, indicating that all three preparations can activate the alternative complement path-
100
Ce/I
re)
.
nl 01
-0.01
0.1
J10
1.0
Component Concentration (mg *y wi /mi serum )
FIG. 1. C3-C9 consumption by S. aureus Hand cell surface fractions in normal human serum. Various weights of intact organisms and fractions were incubated with serum for 60 min at 37°C before determination of C3-C9 consumption.
S'S (a) Normel Serum
(b) C2-eficient Serum
(D)" m Sbuleun-
E 00
c M
05
10 15
Mo51
Mo
105
M
Time (min)
FIG. 2. Kinetics of C3-C9 consumption by S. aureus H PCW and PG in various serum sources. Intact organisms, PCW, and PG were incubated in normal serum (a), C2-deficient serum (b), and immunoglobu-
lin-deficient serum (c) for specified times at a concentration of 1 mg of dry weight/ml of serum. C3-C9 consumption was determined as described in Materials and Methods with a parallel distilled water control included at each time point.
VOL. 20, 1978
STAPHYLOCOCCAL SURFACE AND COMPLEMENT
about 20% of the dry weight of the cell, there would be about five times as many particles or "cell wall" in a PCW preparation as in an equal weight of intact organisms. Thus, if the results were evaluated on an efficiency per particle basis, the intact organisms and PCW would not appear to be greatly different in activity. Consumption of C3-C9 by CCW was less than that by PCW, suggesting that minor cell surface components did not augment C3-C9 consumption. Isolated protein A is capable of activating the alternative and classical pathways of complement (13), and we would perhaps have anticipated CCW to retain this component and be more active than PCW. It is possible that when protein A is in the cell wall it is not as active as when in the isolated state. Also, complement consumption by isolated protein A has been shown to occur in a paradoxical manner where increasing the protein A concentrations beyond an optimal level results in reduced complement consumption-(8, 13). On a weight basis, CM were not as active in complement consumption as intact bacteria. They would seem to be even less active if compared on the basis of the number of organisms required to yield a given weight of CM, since the cell membrane comprises about 10 to 15% of the weight of the staphylococcus. It is unclear to what extent the cell membrane contributes to complement activation by intact organisms, in that the membrane is expected to be somewhat shielded by the cell wall. When complement consumption by PG in normal serum was studied this fraction was clearly not as active as either intact organisms or PCW when equal weights of the preparations were used. The PG was even less active on a particle basis since PG comprises about 50% of cell wall weight and there would, therefore, be about 10 PG particles for every intact organism. In addition, when kinetic studies were performed, complement was activated at a significantly slower rate by PG than by PCW or intact organisms. The essential difference between PCW and PG is the presence of TA and the linkage unit in the former preparations. Thus it appears that TA covalently attached to PG markedly enhances complement activation. In a previous study (P. K. Peterson, et al., J. Clin. Invest., in press) we were unable to demonstrate a significant role for TA in staphylococcal opsonization by the complement system, and PG was the key cell surface component in this regard. Thus, it would appear that the phenomena of bacterial opsonization and complement consumption cannot be automatically equated. Similarly, we have recently found an encapsulated
391
S. aureus strain to significantly activate complement and yet be ineffectively opsonized (lOa). In a kinetic study of chemotactic factor generation in human serum by complement activation, Gallin et al. (4) showed that chemotactic factor generation proceeded more rapidly when mediated by the classical pathway than by the alternative pathway. Those authors concluded that activation by the classical and alternative pathways could be distinguished by kinetic analysis. Since C3-C9 consumption by intact bacteria, PCW, and PG in normal serum was more rapid than in C2-deficient serum, this indicates that both the classical and alternative pathways may be involved in complement activation in normal serum. In serum relatively deficient in antibodies, C3-C9 consumption by the preparations occurred at a significantly slower rate than in normal serum, but somewhat faster than in C2deficient serum. These results suggest that immunoglobulins are necessary to obtain maximal rates of complement activation. Further studies will be needed to delineate the role immunoglobulins play in activation of the classical and/or alternative pathways. Winkelstein and Tomasz (17, 18) evaluated the role of Streptococcus pneumoniae cell surface structures in activating the alternative pathway in C4-deficient guinea pig serum. These authors found that their CCW preparations were less active than PCW and that TA appeared to mediate complement consumption in this serum. S. pneumoniae PG was reported to be inactive in complement consumption. Our results using C2-deficient serum support a significant role for TA in activation of the alternative pathway. However, we found, as have others (2, 11), that staphylococcal PG can activate the alternative pathway, albeit at a slow rate. From these studies it is clear that several S. aureus cell surface components may participate in activation of serum complement. Also, both the classical and alternative pathways can be activated by these components. Further elucidation of the interaction of the staphylococcal cell surface and the serum complement system would appear to be of fundamental importance in understanding host reaction to staphylococcal infection. ACKNOWLEDGMENTS This work was supported by Public Health Service grants AI 6931-11, Al 08821-07, and Al 10704, from the National Institute of Allergy and Infectious Diseases, HL 06314, from the National Heart, Lung and Blood Institute, and by two Grants-in-Aid from the University of Minnesota Graduate School. P. G. Quie is the American Legion Memorial Heart Research Professor. We are grateful to Shirley Hermel for her secretarial help.
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WILKINSON ET AL. LITERATURE CITED
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