Decomplementation Antigen, a Possible Determinant of ...

INFECTION AND IMMUNITY, Jan. 1985, p. 41-46 0019-9567/85/010041-06$02.00/0 Copyright © 1985, American Society for Microbiology

Vol. 47, No. 1

Decomplementation Antigen, a Possible Determinant of Staphylococcal Pathogenicity 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

We report the existence of an extracellular staphylococcal product, designated staphylococcal decomplementation antigen (DA), that causes rapid consumption of early-reacting complement components up to and including C5 in human serum. Complement activation occurs as a consequence of immune complex formation between DA and specific human immunoglobulin G antibodies and proceeds primarily via the classical pathway. The terminal components C7, C8, and C9 are not consumed during the process. Levels of DA production do not correlate with the expression of classical pathogenic factors, such as coagulase, clumping factor, protein A, or at-toxin. DA is a nondialyzable macromolecule eluting in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and displaying an apparent sedimentation coefficient of 3 to 4 S on sucrose density gradients. The molecule is remarkably stable and resists destruction upon boiling for 30 min or by treatment with pronase, lysostaphin, DNase, or RNase. We anticipate that DA protects staphylococci from complement attack through induction of abortive, complement-consuming reactions in the fluid phase.

Phagocytic killing of staphylococci represents the central mechanism of mammalian defense towards these ubiquitous pathogens (8, 10, 13, 19-22). In view of this fact, it is surprising that none of the known and well-studied secreted products which constitute major factors of staphylococcal pathogenicity appear to directly interfere with this host defense system (4, 12). In this communication, we report on the existence of an extracellular substance detectable in cell-free staphylococcal culture supernatants that displays hitherto unrecognized complement-consuming properties. This substance, designated staphylococcal decomplementation antigen (DA), activates the classical pathway through formation of potent, complement-activating immune complexes with specific human immunoglobulin G (IgG). Its production does not strictly correlate with the expression of known pathogenic factors, including coagulase, clumping factor, protein A, and a-toxin. Since complement-dependent opsonisation (18) is a prerequisite for effective phagocytic uptake and killing of most staphylococci (10, 13, 19-22, 25), we anticipate that DA contributes to the pathogenicity of these bacteria through the abortive consumption of C3 in the fluid phase. MATERIALS AND METHODS Staphylococcal strains. Sixteen staphylococcal strains that were characterized with respect to production of coagulase, clumping factor, protein A, and a-toxin were selected from the collection of strains available at the Institute of Microbiology, University of Giessen, Federal Republic of Germany. Assays for clumping factor and protein A were performed as described (14, 15), and the test strains were made available to us through the kindness of W. Schaeg, J. Bruckler, and H. Blobel of the above Institute. Thirty coagulase-positive isolates from human patients were obtained from the routine diagnostic laboratory of our Institute. The bacteria were cultured overnight in Muller-Hinton broth, and the cell-free supernatants were obtained by centrifugation. Assay for decomplementation activity. A pool of reference human serum was diluted 10-fold in veronal-buffered saline *

(25 mM Veronal, 150 mM NaCl [pH 7.4]) containing 0.15 mM Ca2+ and 1.0 mM Mg2+ (VBS) and supplemented with 2.5 mg of Sandoglobulin (Sandoz Laboratories, Nurnberg, Federal Republic of Germany) per ml. Doubling dilutions of culture supernatants were prepared in microtiter plates (Flow Laboratories, Inc., McLean, Va.), 1 volume (50 ,ul) of the human serum pool was added, and incubation was carried out at 37°C for 45 min. Thereafter, 50 plI of a 1% suspension of antibody-coated sheep erythrocytes (EA) was added, and hemolysis was read after 30 min at 37°C. The EA were prepared with antiserum from the Behringwerke, Marburg, Federal Republic of Germany (Ambozeptor, 1:6,000), according to the instructions of the manufacturer. Decomplementation activity was expressed in arbitrary units (AU) and defined as the last dilution of the sample that still inhibited total hemolysis of the cells. In assays performed to demonstrate the IgG dependence of the complement consumption process (see Fig. 4 and 5), the same assay was performed without supplementation of the sera with Sandoglobulin. Assays for consumption of complement components. One volume of bacterial culture supernatant was admixed with one volume of undiluted serum at 37°C. Generally, a serum pool from 20 to 30 healthy adults was used, but individual sera were additionally utilized in certain experiments. Sarmples were withdrawn at intervals of 10, 30, and 60 min for complement assays. C4, C5, C7, C8, and C9 assays were performed by using sera selectively depleted in the component to be titrated. C4-deficient guinea pig serum was a kind gift of D. Bitter-Suermann and U. Hadding (Institute of Medical Microbiology, University of Mainz, Federal Republic of Germany), and C5-depleted human serum was kindly supplied by J. Tschopp (Biochemistry Department, University of Lausanne, Switzerland). Human C7, C8, and C9 were isolated according to published procedures (3, 7, 17), and antisera to these components were raised in rabbits. The IgG fractions of the antisera were obtained by absorption onto and elution with 1 M acetic acid from protein A-Sepharose columns (Pharmacia, Uppsala, Sweden). The IgG-enriched fractions were then coupled to CnBr-activated Sepharose (Pharmacia) following the instructions of the manufacturer.

Corresponding author. 41

42

BHAKDI AND MUHLY

Depletion of

a

human

serum

INFECT. IMMUN.

pool of each complement

AU

component was achieved by chromatographing the serum over the affinity columns at 4°C. Fractions judged to be free of each complement component as assessed by rocket

256 128

immunoelectrophoresis and by hemolytic titrations were pooled. Titrations were performed by preparing doubling dilutions of the sample to be titrated in microtiter plates (VBS buffer). To each well were then added 1 volume (50 Fl) of a 10% solution of the complement-depleted serum in VBS and 50 ,ul of 1% EA. Lysis was read after 60 min at 37°C, and titers were defined as the last dilution giving ¢90% hemolysis in this system. Starting titers were arbitrarily referred to as 100% values, and the titers read were converted to a percentage of the respective starting value. IgG depletion and reconstitution experiments. Human sera were depleted of IgG through a single passage over a protein A-Sepharose column (Pharmacia) at 4°C. The volume ratio of serum to protein A-Sepharose was 1:1, and between 85 and 95% of serum IgG could regularly be removed in this fashion. The IgG was eluted from the columns with 1 M acetic acid in the cold and was immediately reequilibrated in VBS by a passage through Sephadex G-25 (commercially available PD 10 columns; Pharmacia). The IgG fractions were subsequently concentrated by ultrafiltration over Amicon PM 10 membranes (Amicon Corp., Lexington, Mass.) to concentrations of ca. 20 mg/ml and used to reconstitute the IgG-depleted sera. Sephacryl S-300 chromatography and sucrose density gradient centrifugation. Culture supernatants were chromatographed over Sephacryl S-300 (Pharmacia) columns (1 by 60 cm) for obtaining an initial estimate of DA size. Apparent sedimentation coefficients were approximated by centrifugation through 10 to 43% (wt/wt) sucrose density gradients prepared in 10 mM Tris-50 mM NaCl (pH 8.0) at 150,000 x g for 16 h at 4°C (Spinco ultracentrifuge model L2 65 B, rotor type SW65 Ti). A sample of human serum was centrifuged in parallel, and the sedimentation positions of the marker molecules IgM (19S), C3 (9.5S), IgG (7S), and transferrin (5S) were determined by rocket immunoelectrophoresis of the 20 equal fractions harvested from the gradient. Stability of DA. Culture supernatants were subjected to the following treatments: (i) boiling for 30 min, (ii) incubation with pronase (Boehringer, Mannheim, Federal Republic of Germany) or proteinase K (Boehringer) at 50 p.g/ml, (iii) incubation with a mixture of DNase and RNase (Boehringer) at individual enzyme concentrations of 50 p.gIml, or (iv) incubation with 50 jig of lysostaphin (Sigma Chemical Co., Muinich, Federal Republic of Germany) per ml. All enzyme incubations were for 3 h at 37°C. Experiments with staphylococcal a-toxin. This toxin was obtained in its native 3S form as described (5). Electroimmunoassays. Rocket, crossed, and line immunoelectrophoreses were performed as described (9, 23, 24) in 1% agarose (type HSA; Litex Laboratories, Copenhagen, Denmark) with a 0.1 M glycine-0.038 M Tris buffer (pH 8.7). Rabbit antibodies to human C3, IgG, IgM, and transferrin were from Dakopatts Immunoglobulins, Copenhagen, Denmark. RESULTS Demonstration of comp!ement-consuming activity in staphylococcal culture supernatants. Figure 1 shows the typical results obtained by the assay used to screen for decomplementation activity in bacterial culture supernatants. Doubling dilutions of the culture supernatants of four staphylo-

641 32: 16

a

b c d

FIG. 1. Complement consumption induced through the incubation of staphylococcal culture supernatants with human serum. Overnight cultures of four staphylococcal strains (columns a through d) were centrifuged, and the supernatants were serially diluted. Each microtiter well then received 1 volume (50 ,ul) of 10% human serum supplemented with 2.5 mg of IgG (Sandoglobulin) per ml. After 45 min at 37°C, the test was developed by addition of 50 ,u1 of 1% sheep EA to each well. Decomplementation activity was 128, 4, 32, and 4 AU for the tested strains represented by columns a through d, respectively. Low decomplementation activities 2 to 16 AU were found with most staphylococcal strains independent of the presence of DA. Presence of DA in the culture supernatant was always paralleled by decomplementation activities of 32 to 128 AU.

coccal strains (columns a through d) were prepared, 1 volume of 10% human serum supplemented with 2.5 mg of Sandoglobulin per ml was added to each well, and the assays were developed with EA after an incubation of 45 min at 37°C. Low background activities of 2 to 16 AU (e.g., columns b and d) were graded as DA negative, since it was subsequently found that these low activities wcre not derived from the presence of DA. Supernatants containing DA always exhibited decomplementation activities of 32 to 128 AU in this system (columns a and c). TABLE 1. Characterization of 16 staphylococcal strains with respect to production of coagulase, clumping factor, protein A, (a-toxin, and DA Strain

Rd350 Rd382 Rd408 Rd462 Rd475 Rd478 Rd539 Rd565 Rd599 Rd614 Rd661 Rd748 Rd767 Rd784 KalO15 Ka7

Coagulase

Clumping factor

-

-

+

+

-

-

Protein A

a-Toxin

-

+ +

+

-

-

+ + + + + + + + + +

+

-

-

+ +

+

-

-

+

+ -

+

+ +

+ +

+ + + -

DA (AU)

(2) -(8) -(8) + (64) + (32) - (8) + (64) + (128) -(8) -(16) - (8) + (64) - (16) + (64) + (32) -(8)

MODE OF ACTION OF DA

VOL. 47, 1985 0/0

C 7,8

100

A A

80

60

4.0

~~C5

20

20~C 20~~~ \\ ,.C _0 10

20

30

min FIG. 2. Loss of C4 and CS hemolytic titers in pooled human serum through incubation with DA-containing staphylococcal culture supernatant. One volume of serum was incubated with one volume of culture supernatant at 37°C and samples were titrated for individual complement components at the depicted times. Whereas rapid loss of C4 and CS hemolytic titers (expressed in percentages of the starting titers) ensued. C7. C8. and C9 titers remained unchanged, showing the selective consumption of the tested early components up to and including CS during the decomplementation

reaction.

Using this method, we assayed culture supernatants of 30 coagulase- and clumping factor-positive staphylococcal isolates and found DA titers ranging from 32 to 128 AU in all cases. We then examined 16 characterized staphylococcal strains and the results of these investigations are given in Table 1. All coagulase-positive strains were DA positive, and this finding was in accord with the observation made with the 30 coagulase-positive clinical isolates. However, activity was also detected with a few coagulase-negative strains (e.g., strains Rd475 and Rd784). Some strains negative in the expression of one of the other selected pathogenic factors were also DA positive. These findings indicate that DA is not identical to any of these factors. The conclusion is tentative and follows only if the means of recognition of these other factors is at least as sensitive as that for DA. Figures 2 and 3 depict the results of the analysis of individual complement components that were obtained with culture supernatant of strain Rd565. Similar results were obtained with 10 other DA-positive strains tested. The incubation of a serum pool with Muller-Hinton broth led to no consumption of complement. In contrast, incubation with the bacterial culture supernatant caused rapid and complete consumption of C4 and C5; the loss of total hemolytic activity paralleled the loss of C4 titer. C7, C8, and C9, however, remained intact. Crossed immunoelectrophoresis was used to examine the conversion of C3 (Fig. 3). C3 conversion typically exceeded 80% after a 60 min of incubation of a DA-positive culture supernatant with 1 volume of undiluted human serum. DA-mediated decomplementation dependent on the presence of specific human IgG. When a large number of individual human sera were screened for response to DA activity, we occasionally found a serum sample that exhibited only low levels of complement consumption despite incubation

43

with a DA-positive culture supernatant. We utilized these sera to demonstrate that decomplementation was obviously dependent on the presence of specific human IgG antibodies. Two sera, one "positive" and one 'negative' with respect to decomplementation, were selected. Each serum was depleted of IgG through a passage over protein A-Sepharose, and cross-reconstitutions were undertaken with the isolated IgG fractions from both sera. Figure 4A depicts rocket immunoelectrophoreses of the two sera before and after IgG depletion and reconstitutions; the plates were developed with anti-IgG antibodies. Wells a and d contained the positively and negatively reacting sera, respectively. Wells b and e contained the sera after IgG depletion. Wells c and f contained the sera after reconstitution with the homologous IgG fractions. To well g was applied the serum sample of well b cross-reconstituted with IgG of the negative serum, whereas well h received the IgG-depleted serum of well e reconstituted with IgG of the positive serum. It can be seen that IgG levels of the sera before and after reconstitutions were comparable to each other in all cases. The reactions of these serum samples with DA contained in a culture supernatant (strain Rd565) are shown in Fig. 5. Decomplementation activity was assayed for by first serially diluting the supernatant and then adding the serum samples represented by columns a through h. After 45 min at 37°C, EA were given to all wells, and DA titers were read as the last dilutions inhibiting total hemolysis. DA activity was 64 AU as assayed with the positive serum (Fig. 5, column a) and 4 to 8 AU as assayed with the negative serum (column d). Removal of IgG from the sera totally abrogated the

A

(

B

FIG. 3. Conversion of C3 induced by incubation of human serum with 1 volume of DA-containing culture supernatant for 60 min at 37°C. (A) Control serum incubated with sterile Mueller-Hinton culture medium: (B) serum incubated with bacterial supernatant. Note the almost quantitative C3 conversion induced by the latter. First-dimension electrophoresis. right to left. Second-dimension immunoelectrophoresis was performed by using 1 p1 of anti- C3c antibodies per cm2 at 2 V/cm overnight.

44

BHAKDI AND MUHLY

INFECT. IMMUN.

decomplementation effects (columns b and e), and reconstitution of the depleted sera with the homologous IgG fractions restored the decomplementation effects to the original levels (columns c and f). When the originally positive serum was IgG depleted and reconstituted with IgG from the negative serum, poor response to the decomplementation antigen was observed (column g). However, when the negative serum received IgG from the positive serum, a strong decomplementation effect was observed (column h). These results indicated that DA acted by forming complement-activating immune complexes with specific human IgG antibodies and not by nonspecific interaction with immunoglobulins, as is the case with protein A. This contention was borne out by immunoprecipitation experiments and by sucrose density gradient centrifugation analyses. Figure 4B depicts the results of electroimmunoassays. In these analyses, culture supernatant was introduced into a line in the agarose plate, and line immunoelectrophoresis was performed by using Sandoglobulin IgG to develop the immunoplate. As shown in the accompanying paper (1), A

f

ht

.

B -*N-

-0,11

a

b 0 d

FIG. 4. IgG depletion and cross-reconstitution experiments with human serum containing DA antibodies and a serum containing only trace levels of DA antibodies. (A) Rocket immunoelectrophoresis developed with anti-IgG showing the effects of the depletion and reconstitution procedures on total IgG levels. Wells: a, the first, positively reacting serum; b, the same serum after a passage through protein A-Sepharose; c, same as well b after reconstitution with homologous IgG eluted from the protein A well; d, e, and f, analogous samples of the second, negatively reacting serum; g, IgG-depleted serum from well a cross-reconstituted with IgG of serum from well d; h, IgG-depleted serum from well d cross-reconstituted with IgG of serum from well a. All dilution effects occurring during the depletion and reconstitution steps were compensated for by appropriate correction of the sample volumes applied. (B) Demonstration of the presence or absence of precipitating antibodies to DA in the above serum samples a through h. Line immunoelectrophoresis was performed by applying unfractionated culture supernatant of a DA-positive strain in a line and electrophoresing into an agarose gel containing 15 ,ul of pooled human IgG (Sandoglobin) per cm2. The major immunoprecipitate developing as a line (bold arrows) has been identified as DA (1). The faint line precipitate below DA represents another extracellular staphylococcal antigen. Application of serum samples a through h in wells below the Sandoglobulin-containing gel permits direct detection of specific antibodies to DA. These antibodies cause a downward deflection of the line precipitate (small arrows). The specificity of interaction between human IgG with DA is apparent.

a

the major immunoprecipitate developing in this system has been identified as DA (Fig. 4B, arrows). The serum samples represented by wells a through h of Fig. 4A were applied in the wells below the Sandoglobulin-containing agarose (see Fig. 4B), and the presence of antibodies specifically reacting with DA was discernable through the downward deflection of the line immunoprecipitate (for the original description of the depicted method for detecting specific antibodies to an antigen, see reference 11). It can be seen that the positively reacting serum sample a contained specific antibodies to DA, whereas the negatively reacting serum sample d contained only trace amounts of antibody. The immunoplate (Fig. 4B) demonstrates the perfect correlation between presence of specific anti-DA antibodies, either in naturally occurring form or through transfer of IgG, with the decomplementation effects observed in Fig. 5. Sucrose density gradient ultracentrifugation of pooled human IgG with purified DA (1) has subsequently been used to confirm the presence of complement-activating immune complexes sedimenting to high-molecular-weight regions. We have also precipitated the immune complexes with 4% polyethylene glycol 4000 and recovered complement-consuming activity in the precipitates (data not shown). Characterization of DA as a stable macromolecule. DA was nondialyzable. When culture supernatants were chromatographed over Sephacryl S-300, DA activity was detected eluting as a symmetrical peak in a molecular-weight region of 70,000 to 120,000. Upon sucrose density gradient centrifugation, DA activity was observed in the region corresponding to 3 to 4 S. The stability of DA was tested by subjecting culture supernatants to the different treatments detailed above. None of these treatments destroyed the biological activity of DA, which was thus found to be a macromolecule of remarkable chemical stability. Failure of staphylococcal et-toxin to elicit decomplemefitation in serum. Staphylococcal oa-toxin has been reported to elicit complement consumption in human serum (6). Therefore, we added purified toxin to pooled human sera at concentrations of 1 to 100 ,ug/ml. However, we entirely failed to detect significant decomplementation effects of the purified toxin, as assessed by C3 conversion analyses. Thus, DA is not identical to a-toxin. DISCUSSION We detected an extracellular staphylococcal product with remarkable decomplementation potential which, to our knowledge, has not been described before. Within the methodological limitations of the present assays, comparative analyses with 16 selected staphylococcal strains indicate that this factor, which we designated DA, is not identical to coagulase, clumping factor, protein A, or a-toxin. In addition, its resistance towards lysostaphin, proteases, DNase, and RNase indicates that it does not derive from staphylococcal peptidoglycan or nucleic acids, and it may not be a protein. Preliminary data (1) would indicate that the substance represents an extracellular form of teichoic acid. DA activity was detected without exception in culture supernatants of all 30 coagulase-positive clinical isolates tested. We defined the mode of DA action and showed it to function via antibody-dependent complement activation. Human sera selectively depleted of IgG are no longer decomplementated by DA, whereas the effect is restored upon IgG reconstitution. The specificity of DA-IgG interaction is apparent from cross-reconstitution experiments of positively and negatively reacting sera with the respective IgG fractions. The specific reactivity of IgG with DA can also be

MODE OF ACTION OF DA

VOL. 47, 1985

supernatants of even strong producers does not induce marked complement consumption in human serum (unpublished data). In an accompanying paper (1), we report the isolation of DA from bacterial culture supernatants and provide initial evidence that it represents soluble, extracellular teichoic acid. The extraordinary stability and decomplementation potential of DA single it out for a role in suppressing complement-dependent opsonization through

AUI 60

6

50

40

F

30

F

45

abortive complement consumption in the fluid phase and, by inference, for an indirect but effective antiphagocytic function in the human host organism (16).

20 10 a b c d e f

h

FIG. 5. Correlation between presence of specific DA antibodies and elicitation of decomplementation effects by DA. The serum samples a through h of Fig. 4 were incubated with DA-containing culture supernatant, and the decomplementation effects were assessed as described in the legend to Fig. 1. Note the perfect correlation between the elicitation of decomplementation effects and the presence of specific DA antibodies in the samples.

ACKNOWLEDGMENTS We are very grateful to W. Schaeg, J. Bruckler and H. Blobel (Institute of Microbiology, University of Giessen, Federal Republic of Germany) for the gift of the 16 characterized staphylococcal strains tested and for many stimulating and valuable discussions. We thank D. Bitter-Suermann and U. Hadding (Institute of Medical Microbiology, University of Mainz, Federal Republic of Germany) for their kind gift of C4-deficient guinea pig serum, and J. Tschopp (Biochemistry Department, University of Lausanne, Switzerland) for his gift of C5-depleted human serum. These studies were supported by the Deutsche Forschungsgemeinschaft (grant Bh 2/2). LITERATURE CITED 1. Bhakdi, S., and M. Muhly. 1985. Isolation and partial charac-

2.

directly shown by immunoprecipitation in electroimmunoassays. The decomplementation process derives from classical pathway activation, and C4 consumption occurs rapidly. C3 conversion also occurs in the presence of EGTA [ethyleneglycol-bis(3-aminoethyl ether)-N,N-tetraacetic acid] at a slow rate, so that alternative pathway activation seems to proceed to a minor extent in parallel (data not shown). We have not attempted to characterize this process, since it is apparent that the classical pathway represents the major route of activation. The reaction terminates, somewhat surprisingly, at the stage of C5-C6, and we discerned no measurable consumption of C7 through C9 despite full conversion of C3 and total consumption of C5. Thus, although it is generally assumed that generation of C5b in the presence of serum C6 through C9 will automatically be followed by generation of fluid-phase SCSb-9 (2), our present results indicate that this is not invariably true. The factors governing formation of SC5b-9 will require further study. DA is a nondialyzable macromolecule eluting on a Sephacryl column in the molecular-weight region of 70,000 to 120,000 and exhibiting an apparent sedimentation coefficient of 3 to 4S. The heat resistance, as well as the resistance towards pronase, excludes its being identified as staphylococcal ax-toxin. Although the latter has been reported to elicit decomplementation effects in human serum similar to those described in this paper for DA, we failed to confirm this finding with highly purified at-toxin. It is probable that the decomplementation effect of ax-toxin reported previously (6) was derived from contamination of the toxin preparation with DA. The capacity of DA to form potent complement-activating immune complexes with human IgG is remarkable. This property is not shared by a multitude of other extracellular

staphylococcal antigens that are separable from DA by protein purification procedures (1). In addition, we have found that protein A at concentrations found in culture

3. 4.

5.

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amide gel electrophoresis combined with quantitative immunoelectrophoretic methods. Scand. J. Immunol. Suppl. 2:115-121. 12. 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. 13. Peterson, P. K., J. Verhoef, D. Schmeling, and P. G. Quie. 1977. Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. J. Infect. Dis. 136:502-509. 14. Schaeg, W., J. Bruckler, and H. Blobel. 1979. Improved method for the demonstration of Protein A of S. tairweis. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. I Orig. B 245:442-449. 15. Schaeg, W., J. Brucker, 1. Muller, and H. Blobel. 1977. Clumping factor reactions using staphylococci after their extraction with guanidiumchloride. Zentralbl. Bakteriol. Mikrobiol. Hyg.

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