Sep 28, 1984 - Hinton broth, no additives) for 17 to 20 h at 37°C. DA ..... Dulbecco, H. N. Eisen, and H. S. Ginsburg (ed.) ... Wright, A. E., and S. R. Douglas.
Vol. 47, No. 1
INFECTION AND IMMUNITY, Jan. 1985, p. 47-51 0019-9567/85/010047-05$02.00/0 Copyright © 1985, American Society for Microbiology
Isolation and Partial Characterization of Staphylococcal Decomplementation Antigen SUCHARIT BHAKDI* AND MARION MUHLY Institute of Medical Microbiology, University of Giessen, D-6300 Giessen, Federal Republic of Germany Received 19 July 1984/Accepted 28 September 1984
A substance with potent decomplementation activity was isolated from staphylococcal culture supernatants by polyethylene glycol precipitation, DEAE-ion-exchange and Sephacryl chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified substance exhibited all the characteristics of the decomplementation antigen (DA) previously detected in unfractionated culture supernatants. It contained glucosamine and phosphorus and was provisionally identified as extracellular, water-soluble teichoic acid of Staphylococcus aureus. DA was entirely resistent towards the action of proteases, DNase, RNase, or lysostaphin and withstood boiling for 30 min. Its electrophoretic mobility in agarose gels at pH 8.7 was approximately double that of human serum albumin. The molecule eluted in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and sedimented as a symmetrical 3 to 4 S moiety in sucrose density gradients. It migrated near the dye front on 12.5% sodium dodecyl sulfate-polyacrylamide gels and remained undenatured after boiling in sodium dodecyl sulfate. DA formed a symmetrical immunoprecipitate upon crossed immunoelectrophoresis against pooled human immunoglobulin G. It was identified as the major extracellular antigen present in unfractionated S. aureus culture supernatants that is precipitable by naturally occurring human immunoglobulin G antibodies. Immune complexes forming between DA and human immunoglobulin G exhibited an extraordinary capacity to activate the classical complement pathway. Microor nanogram amounts of purified antigen added to antibody-containing human serum effected rapid and complete consumption of C3, C4, and C5. The biochemical and biological properties of DA single out this molecule for an important role in suppressing the opsonizing activity of host complement through induction of abortive complement consumption in the fluid phase. In the accompanying paper (2), we described the existence of a novel extracellular staphylococcal product, designated staphylococcal decomplementation antigen (DA), that appeared to react with specific human immunoglobulin G (IgG) to form immune complexes with unusual complement-activating capacity. We anticipated that this substance represents a hitherto unrecognized determinant of staphylococcal pathogenicity since it probably causes local, abortive complement activation (decomplementation), thus counteracting the opsonic function of complement that is so crucial for effective phagocytosis (7, 9, 11, 14-17, 20). In this paper, we describe the isolation of DA from bacterial culture supernatants, report some unusual features of the molecule, and provisionally identify DA as extracellular teichoic acid. We also identify DA as the major antigen contained in unfractionated staphylococcal culture supernatants that is immunoprecipitated by naturally occurring human IgG antibodies.
lin; Sandoz Laboratories, Nurnberg, Federal Republic of Germany) was retrospectively identified as DA (see below). Bacteria were grown in 2-liter batch cultures (Muller Hinton broth, no additives) for 17 to 20 h at 37°C. DA activity was assayed in the supernatant as described (2) by using a 10% human serum pool supplemented with Sandoglobulin (2.5 mg/ml). Cultures were terminated when DA activities of 64 to 128 arbitrary units were reached. The bacteria were pelleted by centrifugation (Sorvall centrifuge GS-3, rotor HG-4; 7,000 x g, 30 min, 4C), and the supernatants were concentrated ca. 40-fold by continuous ultrafiltration in a Sartorius filatration chamber model 17112 (Sartorius Laboratories, Gottingen, Federal Republic of Germany) by using a membrane type SM 16566 (exclusion limit, 20,000) at 4°C. Thereafter, centrifugation was repeated to remove any residual cells. Solid polyethylene glycol (PEG) 4000 (Merck AG, Darmstadt, Federal Republic of Germany) was added to a final concentration of 20% (wt/vol) to precipitate proteins. DA was quantitatively recovered in the PEG supernatant, which was collected by centrifugation after 60 min of stirring at 4°C (Sorvall centrifuge RC 2B, rotor SS 34). The PEG supernatant was diluted with 3 volumes of distilled water. The conductivity was then 0.8 to 1.0 mS, and the pH was 7 to 7.5. The preparation was directly applied to a 60-ml DEAE-Sephacel column (column dimensions, 2 by 20 cm; Pharmacia, Uppsala, Sweden) equilibrated in 25 mM Veronal-50 mM NaCl (pH 7.0) at 4°C. Fractions (8 ml) were collected at a flow rate of 50 ml/h. After sample application, the column was washed with 90 ml of starting buffer and eluted with a 50- to 500-ml linear NaCl gradient at pH 7.0. The fractions containing DA activity were pooled and concentrated to 7 to 8 ml by ultrafiltration with an Amicon PM 10 membrane and applied to a Sephacryl
MATERIALS AND METHODS We initially selected staphylococcal strain Rd565 for DA isolation since this strain produces little coagulase and protein A but large amounts of DA (2). Subsequent experiments with three Staphylococcus aureus strains confirmed the general applicability of the isolation procedure for obtaining DA from culture supernatants of other DA producers. Isolation protocol. All isolation steps were monitored by assays for DA activity performed as described in the accompanying paper (2). In addition, fused rocket immunoelectrophoresis was used to follow the isolations, since the major precipitate developing with pooled human IgG (Sandoglobu-
*
Corresponding author. 47
48
BHAKDI AND MUHLY
INFECT. IMMUN. It
.
c i)
--o-DA-
0
T.
6.,
..:...,
A
0
_>1
.
:
AU
14
2400-
12
4o
8
12 16 20 24 28 32 36 40 44 48 Fraction
no
FIG. 1. Elution of DA from a DEAE-ion-exchange column at pH 7.0. The 20% PEG supernatant of DA-containing staphylococcal culture supernatant was applied to a DEAE-Sephacel column, and DA was eluted with a linear salt gradient. The molecule was detected by an assay for decomplementation activity (expressed as arbitrary units [AU]) and by fused rocket immunoelectrophoresis of the fractions by using pooled human IgG (Sandoglobulin; 15 p.1/cm2) to develop the immunoplates. DA eluted as a symmetrical peak, and the immunoelectrophoresis shows its separation from two minor, immunoprecipitable contaminants.
S-300 column (2 by 90 cm) equilibrated in 10 mM Tris-50 mM NaCI-7.5 mM NaN3 (pH 8.1). Fractions (8 ml) were collected at a flow rate of 16 ml/h (4°C). DA-containing fractions were pooled, concentrated to 3 to 5 ml, and subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, SDS-polyacrylamide gel slabs were cast as for conventional SDS-PAGE by using a Desaga apparatus (model Havanna) and a previously described gel-buffer system (3). Slots were not formed in the stacking gel. Instead, 1.5-ml samples were loaded across the entire width of the gel. After termination of the electrophoresis, the gel slabs were sectioned into 0.5-cm-wide strips. Each strip was eluted with 10 ml of 10 mM Tris-50 mM NaCl buffer (pH 8.1) overnight. The strips with elution buffer were placed in dialysis bags, and dialysis against the Tris-NaCl buffer was performed during the elution. The DA-containing solutions were decanted, filtered to remove any polyacrylamide gel fragments, and concentrated over Amicon PM 10 membranes. They were finally transferred to saline by a passage over Sephadex G-25 (commercially available PD 10 columns from Pharmacia). The samples could be stored at 4°C for months without loss of activity. DA activity appeared to be lost upon freezing at -20°C. Analytical procedures. Amino acid analyses were performed with the use of an automatic analyzer (Chromakon 500; Kontron Laboratories, Zurich, Switzerland) equipped with an Anacomp 220 computer (Kontron). Phosphate was determined as described (1). Fused rocket, crossed, and
crossed-tandem immunoelectrophoresis were performed as described (5, 8, 18). Assays for DA activity were conducted with a 10% human serum pool diluted in Veronal-buffered saline containing Ca2" and Mg2+ (2) and supplemented with
2.5 mg of Sandoglobulin per ml. Activities were expressed in arbitrary units as described (2). RESULTS Isolation of DA. DA exhibited the advantageous property of not being precipitated by 20% PEG. This greatly facilitated its isolation. DEAE chromatography of PEG supernatants at pH 7.0 led to further purification (Fig. 1). The molecule could be detected by virtue of its decomplementation activity, as well as through the formation of a strong immunoprecipitate with pooled human IgG (Sandoglobulin) antibodies (Fig. 1). DA was found to elute as a symmetrical peak in fractions exhibiting conductivities of 8 to 10 mS. When DA-containing fractions were pooled, concentrated, and chromatographed over Sephacryl S-300, DA eluted in fractions corresponding to a molecular-weight region of 70,000 to 120,000 (Fig. 2). Upon sucrose density gradient centrifugation, an apparent sedimentation coefficient of 3 to 4 S was found. With SDS-PAGE, DA migrated just behind the dye front on 12.5% gels. The molecule was not denatured by boiling in SDS, and immunoprecipitation studies could be carried out after the electrophoresis. For this purpose, the gel strips were soaked in 50% methanol for 3 h to remove the SDS. They were then washed in Tris-NaCl buffer for 15 min and directly laid onto agarose plates containing human IgG. The precipitate developing after immunoelectrophoresis is shown in Fig. 3. DA could thus be isolated in immunologically and functionally intact form. No bands were discerned upon staining of SDS gels loaded with DA, either by conventional protein stains (Coomassie brilliant blue) or with the ultrasensitive silver staining technique.
STAPHYLOCOCCAL DECOMPLEMENTATION ANTIGEN
VOL. 47, 1985 v
it
I/ ,. bIlAK.
AU 5000 4000 3000
49
amounts of DA applied to the peripheral wells (labeled a
through h). Note the strengthening of the immunoprecipitate as the DA concentration was lowered towards the zone of equivalence. Adjustment to this zone was found to be important for elicitation of the complement-activating function. During the course of DA isolations, we always observed prozones in the titrations which were derived from the high concentrations of the antigen. Chemical stability. Isolated DA exhibited the same stability as was found for the antigen in unfractionated culture supernatants. It was not destroyed by treatment with pronase B, lysostaphin, DNase, or RNase, or by boiling in the presence or absence of SDS. The isolated molecule could be stored at 4°C for months without loss of immunoprecipitating and complement-consuming properties. Freezing appeared to reduce its activity and was therefore avoided. DISCUSSION
100( _ _t l L I I 18 20 22 24 26 28 30 Fraction no FIG. 2. Elution of DA on a Sephacryl S-300 column DA-con-
16
romtheDEA coumn erepooed,conentaining frn sctins trated, and chromatographed over Sephacryl S-300. The molecule eluted in a symmetrical peak just behind the elution position of human Ig,G (an arrow denotes the position of the IgG elution). Biological I activity of DA corresponded to the position of the immunoprrecipitate developing in fused rocket immunoelectrophoresis perfoirmed as described in the legend to Fig. 1.
Compaisition. DA preparations contained glucosamine and phosphoirus in approximately equimolar amounts. However trace am ounts of amino acids that may have represented contamin ations were also detected. These findings were taken as preliminary evidence that DA represents an extracellular form of S. aureus teichoic acid (6, 10). The chemical analyses are in a very preliminary stage due to the small amounts of purified material available at oresent. A DA sample containing 30 p.g of glucosamine per ml exhibited a decomplementation titer of 1,000 to 2,000 arbitrary units, i.e., this sample could be diluted over 1,000-fold and still cause loss of hemolytic titer in 10% human serum. It is thus apparent that decomplementation is effected by microgram or nanogram amounts of the antigen. Immunochemical properties. The isolated DA was analyzed by crossed immunoelectrophoresis against pooled human IgG. A sharp, symmetrical immunoprecipitate developed (Fig. 4), and the molecule migrated with very high velocity, approximately twice as fast as human serum albumin at pH 8.7. When an unfractionated culture supernatant of staphylococcal strain Rd565 was similarly analyzed, one major immunoprecipitate developed with the same migration behavior. The identity of this antigen as DA was established by tandem-crossed immunoelectrophoresis (Fig. 4). Analyses with other DA producers subsequently confirmed that this molecule represents the dominant precipitating extracellular antigen of these bacteria. Concentrated DA preparations exhibited marked prozone phenomena, both with respect to immunoprecipitation and complement activation. Figure 5 shows a double diffusion, with human IgG applied to the center well and decreasing
DA was isolated from the culture supernatant of a coagulase-positive staphylococcal strain and was provisionally characterized as a water-soluble macromolecule containing glucosamine and phosphorus. It is thus probably derived from or related to teichoic acid and represents an apparently homogeneous, extracellular form of this moiety. Complement-activating properties of cell wall teichoic acids from staphylococci have been described (16, 19). However, extracellular and water-soluble forms derived from staphylococci have, to our knowledge, not been studied in any detail, and no major biological role has been assigned to these molecules (6, 10). Our data on the physicochemical properties of DA are preliminary. If DA is teichoic acid, its behavior on gel permeation chromatography and sucrose density gradient centrifugation would probably differ markedly from that of a protein. Indeed, the molecule appears moderately large, as judged by Sephacryl chromatography (apparent molecular weight, 70,000 to 120,000), but the sedimentation coefficient in sucrose density gradients is low (3 to 4S). With SDSPAGE, DA also migrates as small molecule, but its strong a negative charge may be causing abnormal migration in this system as well.
..0
0
FIG. 3. Immunoprecipitation of DA after SDS-PAGE. DA recovered from the Sephacryl S-300 column was boiled in SDS and electrophoresed in 12.5% SDS gels (right to left; 0 denotes gel origin). After the electrophoresis, the gel strip was soaked in methanol to remove the SDS, equilibrated in buffer, laid upon an agarose gel, and electrophoresed at right angles into Sandoglobulincontaining agarose. DA yielded a symmetrical precipitate and migrated near the dye front (arrow) in the first-dimension SDSPAGE.
50
BHAKDI AND MUHLY
INFECT. IMMUN.
B
C
4e
0 0 0 0 FIG. 4. Crossed and tandem-crossed immunoelectrophoresis of DA. (A) Purified DA obtained after preparative SDS-PAGE; (B) concentrated, unfractionated culture supernatant of staphylococcal strain Rd565 that was used as the starting material for the DA isolation; (C) tandem-crossed immunoelectrophoresis of purified DA applied in the front well and unfractionated culture supernatant applied in the rear well. All plates were developed with 15 p.l of Sandoglobulin per cm2. First-dimension electrophoresis (right to left) was at 10 V/cm for 25 min. Note the development of a single immunoprecipitate with purified DA, the presence of a major, similarly migrating moiety in the unfractionated culture supernatant, and the identity of these moieties as demonstrated by fusion of the immunoprecipitates in the tandem immunoelectrophoresis. Two minor precipitates (arrows) are discernable, derived from antigens in the culture supernatant. The positions of the application wells are shown (0).
We identified DA as the major precipitable extracellular antigen contained in DA-positive staphylococcal culture supernatants, and it is very surprising that this substance and its unique biological properties have not been detected earlier. All coagulase-positive S. aureus isolates studied to date have been found to produce DA. In contrast, Staphylococcus epidermidis strains appear to be generally DA negative (unpublished data). It is apparent that this finding harbors significant implications with regard to the pathogenicity of staphylococci, which may be related to the difference in chemical structure of teichoic acids produced by these bacteria (6, 10). The extremely fast electrophoretic migration of DA would be consistent with its identity as teichoic acid and might partially explain why the molecule has escaped immunological detection in the past. Thus, DA would migrate out of the agarose gels under the normal conditions used in (crossed) immunoelectrophoresis. The DA-reactive IgG antibodies also have not been previously recognized as major, naturally occurring, precipitating human antibodies to an extracellular staphylococcal product
(4).
We anticipate that DA serves to protect invading bacteria by inducing local, abortive complement consumption in the
h
b
;P'
c
d e
FIG. 5. Prozone phenomenon observed in the immunoprecipitation of DA. Pooled human IgG (Sandoglobulin) was applied to the center well (15 p.l), and 10 .l (a), 8 pl. (b), 6 p.l (c), 5 p.l (d), 4 p.l (e), 2 p.l (f), 1 .l (g), and 0.5 (h) of purified DA was applied to the peripheral wells. A similar prozone phenomenon was observed with respect to the decomplementation activity of DA.
fluid phase. The strategy underlying this process would be straightforward and effective. Judging from the presence of specific antibodies in the majority of human adults (unpublished data), DA is a strong immunogen. If DA is indeed derived from teichoic acid, immunogenicity would be carried in the staphylococcal cell wall (6). The ensuing immunological response and antibody formation in the host would be turned to the advantage of the bacteria. DA produced and liberated into the surroundings of invading staphylococci would bind the antibodies. The generated immune complexes would induce complement activation, probably without fulfilling a defense function for the host. Early components (particularly C3) would be consumed and therefore would not be available to attach to and opsonize the bacteria (12, 13). Biologically active polypeptides released from complement components may enhance inflammatory tissue reactions, but phagocytes attracted to the infected sites would be deprived of the all-important opsonic functions of the complement system. The remarkable stability of DA endows the molecule with special longevity and thus adds a guarantee to its function. Structural studies may provide insights into the nature of immune complexes forming between DA and human IgG, whose capacity to activate human complement is extraordinary. If DA is indeed extracellular teichoic acid, the presense of repeating epitopes may be important. It will also be of great interest to determine whether the decomplementation effect is elicited only by certain classes of extracellular teichoic acid. Immunoprecipitation and complement consumption studies with purified DA revealed a pronounced prozone phenomenon whose recognition is important for construction of the decomplementation experiments. The prozones were not observed at the physiological DA concentrations encountered in the primary culture supernatants. We believe that DA represents a widespread and hitherto unrecognized determinant of staphylococcal pathogenicity. This contention receives support from the unsurprising finding that the substance exerts striking antiphagocytic effects in vitro, and these results will be reported in a separate communication. Although we do not exclude that other extracellular staphylococcal products may express some complement-consuming activities, it appears clear that this function is carried in the main by the DA. Systematic
VOL. 47, 1985
studies with a large number of staphylococcal strains are currently underway to corroborate this contention. Whether or not DA is truly important in pathogenicity may become evident from in vivo studies with selected DA-positive and DA-negative strains in the future. ACKNOWLEDGMENTS We are very grateful to H. Zilg (Behringwerke, Marburg, Federal Republic of Germany) for his valuable help and advice and to W. Schaeg (Institute of Bacteriology, University of Giessen, Federal Republic of Germany) for stimulating discussions. We thank M. Wiesner (Max-Planck Institute of Immunobiology, Freiburg, Federal Republic of Germany) for kindly performing the amino acid analyses. This study was supported by the Deutsche Forschungsgemeinschaft (grant Bh 2/2). LITERATURE CITED 1. Bartlett, G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466-468. 2. Bhakdi, S., and M. Muhly. 1985. Decomplementation antigen, a possible determinant of staphylococcal pathogenicity. Infect. Immun. 47:41-46. 3. Bhakdi, S., J. Tranum-Jensen, and 0. Klump. 1980. The terminal membrane complex of human complement: evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel. J. Immunol. 124:2451-2457. 4. Cohen, J. 0. 1972. Normally occurring antibodies for Staphylococcus aureus, p. 357-363. In J. 0. Cohen (ed.), The staphylococci. Wiley-Interscience, New York. 5. Harboe, N. M. G., and P. J. Svendson. 1983. Fused rocket immunoelectrophoresis. Scand. J. Immunol. Suppl. 10:107-112. 6. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215-257. 7. Koenig, M. G. 1972. The phagocytosis of staphylococci, p. 365-383. In J. 0. Cohen (ed.), The staphylococci. Wiley Interscience, New York. 8. Kr0ll, J. 1973. Tandem-crossed immunoelectrophoresis. Scand. J. Immunol. Suppl. 1:57-59. 9. Leijh, P. C. J., M. T. van den Barselaar, T. L. van Zwet, I.
STAPHYLOCOCCAL DECOMPLEMENTATION ANTIGEN
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Dubbeldeman-Rempt, and R. van Furth. 1979. Kinetics of phagocytosis of S. aureus and E. coli by human granulocytes. Immunology 37:453-465. Morse, S. I. 1980. Staphylococci, p. 623-634. In B. D. Davis, R. Dulbecco, H. N. Eisen, and H. S. Ginsburg (ed.), Microbiology, 3rd ed. Harper & Row, Publishers, New York. Peterson, P. K., J. Verhoef, D. Schmeling, and P. G. Quie. 1977. Kinetics of phagocytosis and bacterial killinig by human polymorphonuclear leukocytes and monocytes. J. Infect. Dis. 136: 502-509. Smith, H. 1983. The elusive determinants of bacterial interference with non-specific host defences. Philos. Trans. R. Soc. Lond. B Biol. Sci. 303:99-113. Stossel, T. P., R. J. Field, J. D. Gitlin, C. A. Alper, and F. S. Rosen. 1975. The opsonic fragment of the third component of human complement. J. Exp. Med. 141:1329-1347. Verbrugh, H. A., P. K. Peterson, B. T. Nguyen, S. P. Sisson, and Y. Kim. 1982. Opsonization of encapsulated S. aureus: the role of specific antibody and complement. J. Immunol. 129: 1681-1687. Verbrugh, H. A., W. C. van Dijk, R. Peters, M. E. van der Tol, and J. Verhoef. 1979. The role of S. aureus cell-wall peptidoglycan, teichoic acid and protein A in the process of complement activation and opsonization. Immunology 37:615-621. Verbrugh, H. A., W. C. van Dijk, M. E. van Erne, R. Peters, P. K. Peterson, and J. Verhoef. 1979. Quantitation of the third component of human complement attached to the surface of opsonized bacteria: opsonin-deficient sera and phagocytosis-resistant strains. Infect. Immun. 26:808-812. Verhoef, J., P. K. Peterson, Y. Kim, L. D. Sabath, and P. G. Quie. 1977. Opsonic requirements for staphylococcal phagocytosis. Immunology 33:191-197. Weeke, B. 1973. Crossed immunoelectrophoresis. Scand. J. Immunol. Suppl. 1:47-59. Wilkinson, B. J., Y. Kim, and P. K. Peterson. 1981. Factors affecting complement activation by Staphylococcus aureus cell walls, their complements, and mutants altered in teichoic acid. Infect. Immun. 32:216-224. Wright, A. E., and S. R. Douglas. 1903. An experimental investigation of the role of the blood fluids in connection with phagocytosis. Proc. R. Soc. Lond. B. Biol. Sci. 72:357-370.
