INFECTION AND IMMUNITY, Oct. 1979, p. 57-63 0019-9567/79/10-0057/07$02.00/0
Vol. 26, No. 1
Chemotaxigenesis by Cell Surface Components of Staphylococcus aureus DAVID J. SCHMELING,l* PHILLIP K. PETERSON,2 DALE E. HAMMERSCHMIDT,2 YOUNGKI KIM,' JAN VERHOEF,2t BRIAN J. WILKINSON,2 3f AND PAUL G. QUIE"3 Departments of Pediatrics, 1 Medicine,2 and Microbiology,3 University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received for publication 11 July 1979
In an attempt to delineate the staphylococcal cell surface components of importance in chemotaxigenesis, we incubated intact Staphylococcus aureus H, crude cell walls, purified cell walls, peptidoglycan, teichoic acid, and cell membranes with human sera. The results reported indicate that both crude cell walls and purified cell walls, as well as peptidoglycan, were potent chemotaxigens. These particles led to the generation in normal human serum of a factor that was chemotactic for human polymorphonuclear leukocytes. Cell wall peptidoglycan and teichoic acid both appeared to play a role in chemotaxigenesis. Kinetic studies employing C2-deficient serum and immunoglobulin-deficient serum revealed that optimal chemotaxigenesis required the presence of an intact classical complement pathway, as well as antibody. Granulocyte aggregometry studies showed that significant levels of C5a were generated in normal serum and that this activated complement component appears to be a major chemotactic factor produced in serum upon interaction with staphylococcal cell wall components.
ymorphonuclear leukocytes (PMNs) through their interaction with human serum and, thus, with the serum C system.
The Staphylococcus is one of the most frequent causes of suppurative infections in humans, and abscess formation is a hallmark of staphylococcal disease. When staphylococci gain access to susceptible tissue, there is an acute inflammatory response involving the directional migration of phagocytic cells into the area of infection (i.e., chemotaxis), a process recognized since ancient times as the formation of "good and laudable pus." Recent studies (10, 13) have shown that cell wall peptidoglycan (PG) of Staphylococcus aureus behaves as a chemotaxigen (an agent that produces a chemotactic factor[s] upon interaction with fresh serum) but not as a chemotaxin (an agent having a direct attractive effect on granulocytes). The staphylococcal cell surface may be viewed as a mosaic network where the cell wall components (PG, teichoic acid [TA], and protein A) and elements of the cell membrane may all be sufficiently exposed to interact with external factors (18, 25) such as the serum complement (C) system (25). The purpose of this investigation was to assess the activities of various S. aureus cell surface components in generation offactors chemotactic for human pol-
MATERIALS AND METHODS
Bacteria and isolation of cell surface fractions. S. aureus H, an organism with a well-established cell wall structure (1), was grown in peptone-yeast extract broth overnight at 370C. The organisms were washed three times in phosphate-buffered saline (PBS) and adjusted to a known concentration via spectrophotometric and pour-plate methods (18); a 1-mg (dry weight)/ml suspension of S. aureus was found to yield 2.7 x 10' colony-forming units per ml. Crude cell walls (CCW), purified cell walls (PCW), PG, TA, and cell membranes were prepared from lateexponential-phase bacteria. The methods of preparation and chemical characterization of these fractions have been described previously (18, 25). Briefly, CCW were isolated by differential centrifugation from organisms disrupted by agitation with glass beads in a Vibrogen Cell Mill (R.H.O. Scientific Inc., Commack, N.Y.). No additional treatments other than water washing were used in the isolation of CCW (25). PCW were prepared by sodium dodecyl sulfate, nuclease, trypsin, and phenol treatments, and PG was isolated from PCW by hot trichloroacetic acid extraction (18). TA was recovered from the trichloroacetic acid supernatant by precipitation with cold ethanol. Cell membranes were isolated by osmotic lysis of lysostaphinPresent address: for State UniLaboratory Microbiology, t induced protoplasts as previously described (25). versity of Utrecht, Utrecht, The Netherlands. The chemical compositions of PCW, PG, and TA t Present address: Department of Biological Sciences, Illiprepared by these methods have been reported prenois State University, Normal, IL 61761. 57
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SCHMELING ET AL.
viously (18) and indicate that the preparations are highly purified. CCW are composed mainly of PG and TA but, in addition, probably contain a small amount of protein A, since proteolytic digestion was not used in their preparation. Previous studies have shown that intact S. aureus H contains protein A (25), and recent findings, indicate that CCW non-specifically binds more '2"I-labeled immunoglobulin than does PCW (K. Schopfer and B. J. Wilkinson, unpublished data), both of which support the presence of protein A in CCW. All cell surface fractions were treated for 30 min in a sonic bath (Branson Sonic Power Co., Danbury, Conn.) before being used in chemotaxigenesis studies since this treatment has been shown to increase the efficacy of PG in C activation (B. J. Wilkinson, Y. Kim, and P. K. Peterson, manuscript in preparation). Enzymatic degradation of PCW and PG was accomplished in the following manner. To a 1-mg/ml PBS suspension of either cell surface fraction was added lysostaphin to a final concentration of 0.5 ,ug/ml. This enzyme specifically dissolves staphylococcal cell walls (21). These mixtures were then incubated at 370C. After the appropriate incubation interval, the lysostaphin was inactivated by heating the samples in boiling water for 10 min. A control sample containing only lysostaphin in PBS was also treated in this fashion and was used to monitor any possible chemotaxigenesis by lysostaphin alone. Serum sources. Normal human serum was collected and pooled from five healthy donors who denied previous staphylococcal infection. C2-deficient serum was obtained from a patient with a genetically determined complete and selective absence of C2 (15) with normal levels of all other C components, including factor B. To block the early classical C pathway component C1, normal serum was treated with ethyleneglycotetraacetic acid in the presence of MgCl2 (MgEGTA) in a 10 mM concentration with respect to serum (7). Immunoglobulin-deficient serum was obtained from a 62-year-old male with common variable immunodeficiency (immunoglobulin G, 73 mg; imnmunoglobulin M, 7 mg; and immunoglobulin A, 3 mg; per 100 ml by laser nephelometry). This serum had normal levels of all C components, except for slightly diminished C1 (96,000 hemolytic units; 126,000 to 369,000 hemolytic units, normal range). All sera were stored in 1.0-ml portions at -700C and thawed immediately before use. Preparation of leukocytes. Heparinized blood (10 U of heparin per ml) was collected from healthy donors and dextran sedimented. The leukocyte-rich plasma was removed with a Pasteur pipette and centrifuged at 160 x g for 10 min. The plasma was decanted, and the leukocyte pellet was washed twice with buffered Hanks balanced salt solution (2). The leukocytes were resuspended in buffered Hanks balanced salt solution to a final concentration of 2 x 106 PMNs/ml. Generation of chemotactic factors in serum. The desired weights of intact organisms or the various cell wall fractions were incubated in serum in an incubator shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 370C and 250 rpm in polypropylene vials (Bio-vials, Beckman Instruments, Inc., Lincolnwood, Ill.) for specified times. Removal of particles from the sera was accomplished by centrifugation at 10,000 x g (4°C) for 15 min. Serum was then carefully
INFECT. IMMUN. transferred into clean, sterile tubes and heated for 30 min at 560C to avoid nonspecific C activation (14). Sera were diluted to 10% concentrations in buffered Hanks balanced salt solution before use in the chemotaxis assay. Chemotaxis. Chemotaxis was assessed by using a modification of a recently described technique (2). Briefly, the lower compartment of a blind-well chamber was filled with the appropriate test serum serving as the chemoattractant, and a membrane filter (pore size, 5 ,sm; Millipore Corp., Bedford, Mass.) was placed into the chamber. Care was taken to avoid bubble accumulation. The top of the chamber was affixed, and excess chemoattractant was removed with a Pasteur pipette before addition of 0.4 ml of the final leukocyte suspension. The chambers were covered to prevent evaporation and then incubated at 370C for 2 h. The filters were removed, fixed, and stained. Cell counts in 10 random high-power fields were used to determine the number of cells having migrated into or through the filter. Each experiment was performed on 3 separate days with leukocytes from three different donors. Generation of chemotactic activity in serum was expressed as a chemotactic index (the ratio of the number of PMNs having migrated into the filter when the attractant was test serum, i.e., serum incubated with one of the test substances, to the number of PMNs having migrated into the filter when the attractant was control serum, i.e., serum incubated with PBS alone). Characterization of the chemotactic factor(s). PMN aggregometry was performed as described previously (3, 4), using serum samples incubated with intact organisms, PCW, or PG (100 ug/ml of serum) or PBS alone for 1 h at 370C as test aggregants of normal and cytochalasin-B-treated granulocytes (4). Aggregating activity was quantitated with an arbitrary scale on which zymosan-activated plasma (ZAP) yielded 100 ZAP units, a 1:2 dilution of ZAP in Hanks balanced salt solution yielded 50 ZAP units, a 1:4 dilution yielded 25 ZAP units, etc. Activation of plasma by zymosan was accomplished by incubation of 1 ml of plasma with 2 mg of zymosan for 60 min at 370C. Chromatography of samples was accomplished in descending, gravity-fed columns of Sephadex G-50 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.), eluting with isotonic PBS (pH 7.4). Artifactual C activation by the column gel was prevented by heating the samples (56°C for 30 min) before chromatography. Chemotactic activity was measured in three elution fractions, less than 10,000, 10,000 to 75,000, and more than 75,000 in molecular weight, as described above for the standard chemotactic assay with undiluted eluent fractions as chemoattractants. Anti-human C5 (goat) antiserum (Behring Diagnostics, Sommerville, N.J.) was freed of complement by heating (56°C for 30 min) and then centrifuged at 10,000 x g for 10 min to remove particulate matter. A 100-gil amount of antiserum was then incubated for 30 min at 37°C with 0.5 ml of the chromatographic fraction to be tested. The resulting preparations were tested for chemotactic and PMN-aggregating activity as described above. RESULTS
Chemotaxigenesis by intact bacteria and
CHEMOTAXIGENESIS BY S. AUREUS CELL WALLS
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cell surface fractions in normal human serum. Intact bacteria and cell surface fractions, in weights from 0.01 to 1.0 mg, were added individually to 1-ml portions of normal human serum and incubated for 1 h at 370C. Generation of chemotactic activity due to activation of the serum C system (23) by each of these components was expressed as a ratio relative to control serum incubated with PBS. All of the materials tested generated some chemotactic activity at doses as low as 0.1 mg/ml of serum, and, with the exception of intact bacteria, all particles generated significant chemotactic activity at concentrations of 0.01 mg/ml of serum (Fig. 1). None of the cell wall components generated chemotactic activity in serum which had been heat inactivated (560C for 30 min) before incubation with each of the test particles (data not shown). These results indicate that, when compared on an equal-weight basis, CCW, PCW, and PG were approximately equal in their potency as chemotaxigens, and they were more active than intact organisms or cell membranes at doses of 0.1 and 0.01 mg/ml of serum. At higher concentrations (0.5 and 1 mg/ml of serum), however, both intact organisms and cell membranes were capable of significant chemotaxigenesis. The equivalent chemotaxigenesis engendered by the CCW, PCW, and PG preparations would seem to indicate that neither cell wall protein (present in CCW) nor TA (present in both CCW and PCW preparations) plays a significant role in chemotaxigenesis and that PG is the major chemotaxigen. However, since PG makes up 16.0
59
only about half of the cell wall weight (25), there are about two PG particles for every CW particle when these preparations are compared on an equal-weight basis. Therefore, PCW and PG preparations were assessed on both an equalweight and an equal-particle basis. Also, kinetic studies were performed to further evaluate the potencies of these chemotaxigens. These studies were undertaken by incubating 0.1 mg each of intact organisms, CCW, and PCW and 0.1 and 0.05 mg of PG in normal serum for 1, 5, 15, and 60 min and determining the relative rates of generation of chemotactic activity. Figure 2 shows that CCW and PCW elicit a significantly more rapid generation of chemotactic activity than do either PG particles or intact S. aureus H, which at this weight is only minimally chemotaxigenic. Figure 2 also shows that PCW is a more potent chemotaxigen than is PG when compared on a particle-per-particle rather than an equal-weight basis. The faster rate of chemotaxigenesis elicited by CCW and PCW may suggest a role for TA in the generation of chemotactic factors. Given a full hour of incubation time, however, equal weights of PG, CCW, and PCW were essentially equal in the absolute amounts of chemotactic activity generated. Chemotaxigenesis by PCW and PG in other serum sources. To delineate which complement pathway(s) is utilized in the generation of chemotactic activity and to evaluate the role of immunoglobulins in this process, we incusbated PCW and PG particles in normal serum and in MgEGTA-chelated, C2-deficient, and immunoglobulin-deficient sera for 1, 5, 15, and 60 min. As shown in Fig. 3, when these particles were incubated for 60 min in normal serum,
12.0 10.0
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Crude Cell Walls (CCW)
0.5 0.11 Purified Cell Wolls
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Peptidoglycon (PG)
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(PCW)
FIG. 1. Dose response of chemotaxigenesis. All particles were tested at the indicated weights by incubation in 1 ml of normal serum for 60 min at
370C. Values are averages of three experiments with indicated ranges.
60
Incubation Time (minutes)
I06.50.10.
FIG. 2. Kinetics of chemotaxigenesis elicited by intact organisms and the major cell surface fractions. Intact S. aureus H, CCW, and PCW, each at 100 pg, and PG at 50 and 100 pg were exposed to normal serum for the indicated times, and the chemotactic activity was measured. Values are averages of three
experiments.
60
INFECT. IMMUN.
SCHMELING ET AL.
~~~~~~~~~~~~~~~~def~clent
041
-10.0
2 80 0
E
6.0 4.0
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5
15
60
5
15
60
Incubation Time (minutes) FIG. 3. Comparison of the kinetics of chemotaxigenesis elicited by PCW(A) or PG (B) in normal, MgEGTAchelated, C2-deficient, or immunoglobulin-deficient serum. A 100-pug amount of either PCW or PG was added to 1 ml of each serum. Chemotaxigenesis was measured after incubation at 37'C for the indicated times. Values represent averages of three experiments.
MgEGTA-chelated serum, and C2-deficient serum, approximately equal amounts of chemotactic activity were generated. When the rates of chemotaxigenesis in these sera were compared, chemotactic activity was generated at a somewhat slower rate in C2-deficient serum than in either normal serum or MgEGTA-chelated serum. Chemotactic activity was generated at a faster rate by PCW than by PG in these sera. The results with C2-deficient serum suggest that chemotaxigenesis can occur in the absence of the classical C pathway, i.e., by alternative pathway activation, although the rate of chemotaxigenesis is decreased in the absence of the classical pathway. When the classical C pathway is blocked by MgEGTA, however, the rate of chemotaxigenesis is relatively unimpaired. The discrepancy between the rate of chemotaxigenesis in the MgEGTA-chelated and C2-deficient sera might possibly be explained by fluid-phase activation of alternative pathway components by Mg2+ (5, 6). Immunoglobulin-deficient serum served as a relatively poor source for chemotaxigenesis by both PCW and PG (Fig. 3). This finding suggests that antibody is necessary for the optimal generation of chemotactic activity by both particles. Effect of solubilization of PCW and PG on chemotaxigenesis in normal human serum. To determine whether enzymatic alteration of particle size would influence the ability of PCW or PG to induce chemotactic activity in
normal serum, we subjected these particles to treatment with the cell wall lytic enzyme lysostaphin for various periods of times. As shown in Fig. 4, this treatment eventually resulted in solubilization of the PCW and PG preparations. The duration of enzymatic treatment of PCW and PG had a marked influence on the chemotactigenic efficiencies of these particles, with a marked reduction in the abilities of these preparations to generate chemotactic activity paralleling the reduction in optical density of the two suspensions. These results indicate that larger particles are more effective than smaller fragments in generating chemotactic activity in serum. In view of the specificity of lysostaphin, the finding that chemotaxigenic activity was abolished upon extensive lysostaphin digestion of PCW and PG indicates that the activities of the intact particles are not due to contaminating material. When isolated TA, a water-soluble cell wall component, was tested for its ability to generate chemotactic activity in normal serum, it was found to be a relatively ineffective chemotaxigen. When 100 1Lg of TA was incubated in normal serum for 60 min, the chemotactic index was 3.6. However, TA was a more potent chemotaxigen than either the PCW or PG preparation that had been solubilized by 2-h lysostaphin treatment. The chemotaxigenic activity of TA may explain the greater potency of PCW as a chemotaxigen compared with PG.
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CHEMOTAXIGENESIS BY S. AUREUS CELL WALLS
0-
8
-0.42 6.0
(
4.0
E
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Lysostaphin Treatment (minutes) FIG. 4. Effect of lysostaphin treatment of PCW
and PG on chemotaxigenesis. PCW and PG were subjected to the cell wall lytic enzyme lysostaphin for the indicated times, and these enzymatic digests were incubated in normal serum for 15 min. Dashed lines indicate the optical density of the lysostaphin-treated particles, and solid lines indicate chemotaxigenic activity.
Characterization of the chemotactic factor generated in normal serum. To determine the nature of the chemotactic factor(s) produced in serum by staphylococcal cell wall components, we performed experiments with granulocyte aggregometry. In previous studies,
PMN-aggregating activity in serum or plasma has been attributed to the presence of C5a (3, 11, 17). Normal serum incubated separately with 100 ,tg of intact organisms, PG, or PCW was compared for PMN-aggregating activity with ZAP. All three particles were capable of rendering serum a potent aggregant of normal granulocytes; PCW and PG each generated 90 to 110 ZAP units of activity when incubated in serum, whereas intact organisms yielded 40 to 55 ZAP units in comparable incubations. These results closely paralleled the chemotactic activity exhibited by these sera. When serum activated with 100 jug of PG was subjected to Sephadex G-50 gel filtration, a single peak of chemotactic and PMN-aggregating activity appeared at a molecular weight of about 15,000, similar to the reported molecular weight of C5a (23). Three molecular weight fractions of equal volume were collected: a low-molecularweight fraction (less than 10,000) which yielded a chemotactic index of 0.9, a middle-molecularweight fraction (10,000 to 75,000) which displayed peak PMN-aggregating activity (22 ZAP units) and gave a chemotactic index of 5.9, and a high-molecular-weight fraction (more than 75,000) which displayed no aggregating activity and yielded a chemotactic index of only 0.6 (indices given are the mean values of three separate experiments).
61
Both the chemotactic and PMN-aggregating capacities of activated serum were markedly inhibited by incubation with anti-C5 antiserum, with the chemotactic index of the middle-molecular-weight fraction being reduced from 5.9 to 1.7 and the aggregating activity being reduced from 22 to 3 ZAP units. These findings indicate that C5a is an important chemotactic factor and may be the major chemotactically active fragment generated by incubation of the various cell wall components in human serum.
DISCUSSION Staphylococcal infections are generally characterized by the formation of pus, and this feature suggests the active production of chemotactic factors by the staphylococcus in vivo. Results of recent studies by Russell and colleagues (20) have implicated the serum C system as the chief mediator of neutrophil chemotactic responsiveness to staphylococcal infection. These investigators have concluded that the attraction of neutrophils by factors released from staphylococci and acting directly as chemotaxins is probably trivial. It was the purpose of the current study to evaluate the activities of various staphylococcal cell surface components as chemotaxigens in human serum, i.e., as substances which are capable of generating chemotactic activity in serum.
The results presented demonstrate that all S. aureus H cell surface components tested were capable of functioning as chemotaxigens. On an equal-weight basis, CCW, PCW, and PG were found to be more potent chemotaxigens than were intact organisms or cell membranes. As the intact organisms and the CCW preparation were not significantly more active than the PCW or PG preparations, it would appear that cell wallassociated protein A, which is present in the intact organisms and is anticipated to be present in the CCW but is not found in PCW or PG (25), does not contribute in a major way to the generation of chemotactic activity in normal serum. Other investigators, however, have found that when protein A is isolated from the cell wall, it is capable of activating both the classical and alternative C pathways (19,24) and of generating chemotactic activity (12). It is possible that in the isolated state nonspecific binding of Fc fragments of immunoglobulin to protein A and formation of immune complexes are facilitated and that steric hindrances to these phenomena may occur when protein A is cell wall associated. Miller (16) and Gallin et al. (8) have recently emphasized the importance of studying the kinetics of chemotaxigenesis, as it has been shown that whereas activation of the classical C path-
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SCHMELING ET AL.
way results in the rapid generation of chemotactic activity, activation of the alternative C pathway is typically characterized by a 15- to 30-min delay before the appearance of chemotactic activity. In the present investigation, kinetic studies revealed that when normal serum was used, CCW and PCW generated chemotactic activity at a more rapid rate than did PG. Also, when PCW and PG were compared on an equal-particle, rather than on an equal-weight, basis as chemotaxigens, PCW was found to be more active than PG. As the major difference between PCW and PG is the absence of TA and its linkage unit in the PG preparation, it appears that when TA is covalently linked to PG the rate of chemotaxigenesis is enhanced. Kinetic studies also revealed that although intact bacteria, PCW, and PG were capable of generating chemotactic activity in the absence of an intact classical C pathway, the rate of chemotaxigenesis was significantly slower in C2deficient serum than in normal serum, suggesting a role for the early components of the classical pathway in this process. An apparent contradiction to this proposal, however, was provided by the finding that the rate of chemotaxigenesis was similar in normal serum and in MgEGTA-chelated serum. Although the explanation for the discrepant findings with C2-deficient serum and MgEGTA-chelated serum has not been established, it is possible that spontaneous fluid-phase activation of alternative pathway components by Mg2e may have artifically enhanced the rate of chemotaxigenesis in MgEGTA-chelated serum, as has been suggested by other investigators (5, 6) using different experimental systems. In serum relatively deficient in immunoglobulins, chemotaxigenesis by the different cell wall preparations developed at a slower rate and to a significantly lesser extent than in normal human serum. These results suggest that immunoglobulins are required for efficient chemotaxigenesis by the staphylococcal cell wall. In support of this conclusion, Grov and Sveen (10) have reported recently that antibodies are necessary for effective chemotaxigenesis by PG in mouse serum.
Experiments with lysostaphin-degraded PCW and PG led to the conclusion that small soluble fragments of the cell wall are relatively inefficient chemotaxigens. Isolated TA, which is water soluble, was found to be a less potent chemotaxigen that were particulate PCW and PG; however, it was more effective than were heavily degraded PCW and PG. These results closely parallel those obtained in C consumption studies recently completed in this laboratory (Wilkinson
INFECT. IMMUN.
et al., manuscript in preparation). That a cell wall particle must be of a certain minimal size for optimal chemotaxigenesis is supported by the work of Grov and Sween (10), who found the critical size of PG fragments to be about 2,000 molecular weight. Snyderman et al. (22) have demonstrated that the major neutrophil chemotactic factor resulting from in vitro C activation is a heat-stable, small-molecular-weight (17,500) cleavage product of the fifth component of complement, designated C5a. Craddock and colleagues (3), as well as O'Flaherty and his co-workers (17), have shown recently that the chemotactic fragment of C5 is capable of inducing PMN aggregation and swelling and that the chemotactic activity of this preparation paralleled its aggregating activity. Gallin and colleagues (9) demonstrated that exposure of granulocytes to preparations of C5a resulted in a decrease in the net negative surface charge of the cell and suggested that a decrease in surface charge is a prerequisite for directed locomotion. Results from the present study, using granulocyte aggregometry as a measure of C5a (3), confirm the importance of this factor in promoting chemotaxis. C5a was found to be present in serum activated by intact bacteria, PCW, and PG. Evidence that C5a is the fragment responsible for chemotactic activity was provided by the observation that anti-C5 antiserum inhibited the ability ofthese activated sera to function as chemoattractants. In conclusion, these in vitro studies demonstrate that several S. aureus cell wall components may be involved in the generation of chemotactic factors in serum. In vivo, these same cell wall components might be expected to contribute as chemotaxigens, leading to the formation of "laudable pus." The optimal generation of the chemotactically active cleavage product, C5a, by these staphylococcal cell wall components appears to depend upon the participation of immunoglobulins and the classical and alternative C pathways. ACKNOWLEDGME14ENTS This work was supported by Public Health Service grants AI 08821, A106931, and HL19725 from the National Institutes of Health and grants-in-aid from the Minnesota Medical Foundation. P.G.Q. is an American Legion Heart Research Professor.
LITERATURE CITED 1. Archibald, A. R. 1972. The chemistry of staphylococcal cell walls, p. 75-109. In J. 0. Bohen (ed.), The staphylococci. Wiley Interscience, New York. 2. Cates, K. L, C. E. Ray, and P. G. Quie. 1978. Modified Boyden chamber methods for measuring polymorphonuclear leukocyte chemotasxis, p. 67-71. In J. I. Gallin (ed.), Leukocyte chemotaxis. Raven Press, New York.
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3. Craddock, P. R., D. E. Hammerschmidt, J. G. White, A. P. Dalmasso, and H. S. Jacob. 1977. Complement (C5a)-induced granulocyte aggregation in vitro: a possible mechansim of complement-mediated leukostasis and leukopenia. J. Clin. Invest. 60:261-264. 4. Craddock, P. R., J. G. White, and H. S. Jacob. 1978. Potentiation of complement (C5a)-induced granulocyte aggregation by cytochalasin-B. J. Lab. Clin. Med. 91: 490-499. 5. Des Prez, R. M., C. S. Bryan, J. Hawiger, and D. G. Colley. 1975. Function of the classical and alternative pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgCl2-ethylene glycol tetraacetic acid. Infect. Immun. 11:1235-1243. 6. Fine, D. P. 1977. Comparison of ethyleneglycoltetraacetic acid and its magnesium salt as reagents for studying alternative complement pathway function. Infect. Immun. 16:124-128. 7. Forsgren, A., R. H. McLean, A. F. Michael, and P. G. Quie. 1975. Studies of the alternate pathway in chelated serum. J. Lab. Clin. Med. 85:904-912. 8. 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 pathway. Clin. Immunol. Immunopathol. 3:334-346. 9. Gallin, J. I., J. R. Durocher, and A. P. Kaplan. 1975. Interaction of chemotactic factors with the cell surface. I. Chemotactic factor induced changes in human granulocyte surface charge. J. Clin. Invest. 55:967-974. 10. Grov, A., and K. Sveen. 1978. Induction of leukochemotaxis by peptidoglycan of Staphylococcus aureus. Acta Pathol. Microbiol. Scand. Sect. B 86:375-378. 11. Hammerschmidt, D. E., P. R. Craddock, J. McCullough, R. S. Kronenberg, A. P. Dalmasso, and H. S. Jacob. 1978. Complement activation and pulmonary leukostasis during nylon fiber filtration leukopheresis. Blood 51:721-730. 12. Harvey, R. L, G. Kronvall, G. M. Troup, R. F. Anderson, and R. C. Williams, Jr. 1970. Chemotaxis of polymorphonuclear leukocytes by protein A of the staphylococcus. Proc. Soc. Exp. Biol. Med. 135:453456. 13. Heymer, B., and E. T. Rietschel. 1977. Biological properties of peptidoglycans, p. 344-349. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 14. Keller, H. V., M. W. Hess, and H. Cottier. 1974. The in
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vitro assessment of leukocyte chemotaxis. Antibiot. Chemother. (Washington, D.C.) 19:112-125. 15. Kim, Y., P. S. Friend, L G. Dresner, E. G. Yunis, and A. F. Michael. 1977. Inherited deficiency of the second component of complement (C2) with membranoproliferative glomerulinephritis. Am. J. Med. 62:765-771. 16. Miller, M. E. 1975. Pathology of chemotaxis and random mobility. Semin. Hematol. 12:59-82. 17. O'Flaherty, J. T., D. L. Kreutzer, and P. A. Ward. 1978. Chemotactic factor influences on the aggregation, swelling, and foreign surface adhesiveness of human leukocytes. Am. J. Pathol. 90:537-550. 18. Peterson, P. K., B. J. Wilkinson, Y. Kim, D. Schmeling, S. D. Douglas, P. G. Quie, and J. Verhoef. 1978. The key role of peptidoglycan in the opsonization of Staphylococcus aureus. J. Clin. Invest. 61:597-609. 19. Pryjma, J., K. Pryima, A. Grov, and P. B. Heczko. 1976. Immunological activity of staphylococcal cell wall antigens, p. 873-881. In J. Jeljasweicz (ed.), Staphylococci and staphylococcal disease. Gustav Fisher Verlag, New York. 20. Russell, R. J., P. C. Wilkinson, R. J. Mchnroy, S. McKay, A. C. McCartney, and J. P. Arbuthnott. 1976. Effects of staphylococcal products on locomotion and chemotaxis of human blood neutrophils and monocytes. J. Med. Microbiol. 8:433-449. 21. Schindler, C. A., and V. T. Schuhardt. 1964. Lysostaphin: a new bacteriolytic agent for the staphylococcus. Proc. Natl. Acad. Sci. U.S.A. 51:414-421. 22. Snyderman, R., H. S. Shin, J. K. Phillips, H. Gewurz, and S. E. Mergenhagen. 1969. Polymorphonuclear leukocyte chemotactic activity in rabbit serum and guinea pig serum treated with immune complexes: evidence for C5a as the major chemotactic factor. Infect. Immun. 1:521-525. 23. Snyderman, R., H. S. Shin, J. K. Phillips, H. Gewurz, and S. E. Mergenhagen. 1969. A neutrophil chemotactic factor derived from C'5 upon interaction of guinea pig serum with endotoxin. J. Immunol. 103:413-422. 24. Stilenheim, G., 0. Gotz, 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. 25. Wilkinson, B. J., Y. Kim, P. K. Peterson, P. G. Quie, and A. F. Michael. 1978. Activation of complement by cell surface components of Staphylococcus aureus. Infect. Immun. 20:388-392.
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Chemotaxigenesis by Cell Surface Components of Staphylococcus aureus DAVID J. SCHMELING,l* PHILLIP K. PETERSON,2 DALE E. HAMMERSCHMIDT,2 YOUNGKI KIM,' JAN VERHOEF,2t BRIAN J. WILKINSON,2 3f AND PAUL G. QUIE"3 Departments of Pediatrics, 1 Medicine,2 and Microbiology,3 University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received for publication 11 July 1979
In an attempt to delineate the staphylococcal cell surface components of importance in chemotaxigenesis, we incubated intact Staphylococcus aureus H, crude cell walls, purified cell walls, peptidoglycan, teichoic acid, and cell membranes with human sera. The results reported indicate that both crude cell walls and purified cell walls, as well as peptidoglycan, were potent chemotaxigens. These particles led to the generation in normal human serum of a factor that was chemotactic for human polymorphonuclear leukocytes. Cell wall peptidoglycan and teichoic acid both appeared to play a role in chemotaxigenesis. Kinetic studies employing C2-deficient serum and immunoglobulin-deficient serum revealed that optimal chemotaxigenesis required the presence of an intact classical complement pathway, as well as antibody. Granulocyte aggregometry studies showed that significant levels of C5a were generated in normal serum and that this activated complement component appears to be a major chemotactic factor produced in serum upon interaction with staphylococcal cell wall components.
ymorphonuclear leukocytes (PMNs) through their interaction with human serum and, thus, with the serum C system.
The Staphylococcus is one of the most frequent causes of suppurative infections in humans, and abscess formation is a hallmark of staphylococcal disease. When staphylococci gain access to susceptible tissue, there is an acute inflammatory response involving the directional migration of phagocytic cells into the area of infection (i.e., chemotaxis), a process recognized since ancient times as the formation of "good and laudable pus." Recent studies (10, 13) have shown that cell wall peptidoglycan (PG) of Staphylococcus aureus behaves as a chemotaxigen (an agent that produces a chemotactic factor[s] upon interaction with fresh serum) but not as a chemotaxin (an agent having a direct attractive effect on granulocytes). The staphylococcal cell surface may be viewed as a mosaic network where the cell wall components (PG, teichoic acid [TA], and protein A) and elements of the cell membrane may all be sufficiently exposed to interact with external factors (18, 25) such as the serum complement (C) system (25). The purpose of this investigation was to assess the activities of various S. aureus cell surface components in generation offactors chemotactic for human pol-
MATERIALS AND METHODS
Bacteria and isolation of cell surface fractions. S. aureus H, an organism with a well-established cell wall structure (1), was grown in peptone-yeast extract broth overnight at 370C. The organisms were washed three times in phosphate-buffered saline (PBS) and adjusted to a known concentration via spectrophotometric and pour-plate methods (18); a 1-mg (dry weight)/ml suspension of S. aureus was found to yield 2.7 x 10' colony-forming units per ml. Crude cell walls (CCW), purified cell walls (PCW), PG, TA, and cell membranes were prepared from lateexponential-phase bacteria. The methods of preparation and chemical characterization of these fractions have been described previously (18, 25). Briefly, CCW were isolated by differential centrifugation from organisms disrupted by agitation with glass beads in a Vibrogen Cell Mill (R.H.O. Scientific Inc., Commack, N.Y.). No additional treatments other than water washing were used in the isolation of CCW (25). PCW were prepared by sodium dodecyl sulfate, nuclease, trypsin, and phenol treatments, and PG was isolated from PCW by hot trichloroacetic acid extraction (18). TA was recovered from the trichloroacetic acid supernatant by precipitation with cold ethanol. Cell membranes were isolated by osmotic lysis of lysostaphinPresent address: for State UniLaboratory Microbiology, t induced protoplasts as previously described (25). versity of Utrecht, Utrecht, The Netherlands. The chemical compositions of PCW, PG, and TA t Present address: Department of Biological Sciences, Illiprepared by these methods have been reported prenois State University, Normal, IL 61761. 57
58
SCHMELING ET AL.
viously (18) and indicate that the preparations are highly purified. CCW are composed mainly of PG and TA but, in addition, probably contain a small amount of protein A, since proteolytic digestion was not used in their preparation. Previous studies have shown that intact S. aureus H contains protein A (25), and recent findings, indicate that CCW non-specifically binds more '2"I-labeled immunoglobulin than does PCW (K. Schopfer and B. J. Wilkinson, unpublished data), both of which support the presence of protein A in CCW. All cell surface fractions were treated for 30 min in a sonic bath (Branson Sonic Power Co., Danbury, Conn.) before being used in chemotaxigenesis studies since this treatment has been shown to increase the efficacy of PG in C activation (B. J. Wilkinson, Y. Kim, and P. K. Peterson, manuscript in preparation). Enzymatic degradation of PCW and PG was accomplished in the following manner. To a 1-mg/ml PBS suspension of either cell surface fraction was added lysostaphin to a final concentration of 0.5 ,ug/ml. This enzyme specifically dissolves staphylococcal cell walls (21). These mixtures were then incubated at 370C. After the appropriate incubation interval, the lysostaphin was inactivated by heating the samples in boiling water for 10 min. A control sample containing only lysostaphin in PBS was also treated in this fashion and was used to monitor any possible chemotaxigenesis by lysostaphin alone. Serum sources. Normal human serum was collected and pooled from five healthy donors who denied previous staphylococcal infection. C2-deficient serum was obtained from a patient with a genetically determined complete and selective absence of C2 (15) with normal levels of all other C components, including factor B. To block the early classical C pathway component C1, normal serum was treated with ethyleneglycotetraacetic acid in the presence of MgCl2 (MgEGTA) in a 10 mM concentration with respect to serum (7). Immunoglobulin-deficient serum was obtained from a 62-year-old male with common variable immunodeficiency (immunoglobulin G, 73 mg; imnmunoglobulin M, 7 mg; and immunoglobulin A, 3 mg; per 100 ml by laser nephelometry). This serum had normal levels of all C components, except for slightly diminished C1 (96,000 hemolytic units; 126,000 to 369,000 hemolytic units, normal range). All sera were stored in 1.0-ml portions at -700C and thawed immediately before use. Preparation of leukocytes. Heparinized blood (10 U of heparin per ml) was collected from healthy donors and dextran sedimented. The leukocyte-rich plasma was removed with a Pasteur pipette and centrifuged at 160 x g for 10 min. The plasma was decanted, and the leukocyte pellet was washed twice with buffered Hanks balanced salt solution (2). The leukocytes were resuspended in buffered Hanks balanced salt solution to a final concentration of 2 x 106 PMNs/ml. Generation of chemotactic factors in serum. The desired weights of intact organisms or the various cell wall fractions were incubated in serum in an incubator shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 370C and 250 rpm in polypropylene vials (Bio-vials, Beckman Instruments, Inc., Lincolnwood, Ill.) for specified times. Removal of particles from the sera was accomplished by centrifugation at 10,000 x g (4°C) for 15 min. Serum was then carefully
INFECT. IMMUN. transferred into clean, sterile tubes and heated for 30 min at 560C to avoid nonspecific C activation (14). Sera were diluted to 10% concentrations in buffered Hanks balanced salt solution before use in the chemotaxis assay. Chemotaxis. Chemotaxis was assessed by using a modification of a recently described technique (2). Briefly, the lower compartment of a blind-well chamber was filled with the appropriate test serum serving as the chemoattractant, and a membrane filter (pore size, 5 ,sm; Millipore Corp., Bedford, Mass.) was placed into the chamber. Care was taken to avoid bubble accumulation. The top of the chamber was affixed, and excess chemoattractant was removed with a Pasteur pipette before addition of 0.4 ml of the final leukocyte suspension. The chambers were covered to prevent evaporation and then incubated at 370C for 2 h. The filters were removed, fixed, and stained. Cell counts in 10 random high-power fields were used to determine the number of cells having migrated into or through the filter. Each experiment was performed on 3 separate days with leukocytes from three different donors. Generation of chemotactic activity in serum was expressed as a chemotactic index (the ratio of the number of PMNs having migrated into the filter when the attractant was test serum, i.e., serum incubated with one of the test substances, to the number of PMNs having migrated into the filter when the attractant was control serum, i.e., serum incubated with PBS alone). Characterization of the chemotactic factor(s). PMN aggregometry was performed as described previously (3, 4), using serum samples incubated with intact organisms, PCW, or PG (100 ug/ml of serum) or PBS alone for 1 h at 370C as test aggregants of normal and cytochalasin-B-treated granulocytes (4). Aggregating activity was quantitated with an arbitrary scale on which zymosan-activated plasma (ZAP) yielded 100 ZAP units, a 1:2 dilution of ZAP in Hanks balanced salt solution yielded 50 ZAP units, a 1:4 dilution yielded 25 ZAP units, etc. Activation of plasma by zymosan was accomplished by incubation of 1 ml of plasma with 2 mg of zymosan for 60 min at 370C. Chromatography of samples was accomplished in descending, gravity-fed columns of Sephadex G-50 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.), eluting with isotonic PBS (pH 7.4). Artifactual C activation by the column gel was prevented by heating the samples (56°C for 30 min) before chromatography. Chemotactic activity was measured in three elution fractions, less than 10,000, 10,000 to 75,000, and more than 75,000 in molecular weight, as described above for the standard chemotactic assay with undiluted eluent fractions as chemoattractants. Anti-human C5 (goat) antiserum (Behring Diagnostics, Sommerville, N.J.) was freed of complement by heating (56°C for 30 min) and then centrifuged at 10,000 x g for 10 min to remove particulate matter. A 100-gil amount of antiserum was then incubated for 30 min at 37°C with 0.5 ml of the chromatographic fraction to be tested. The resulting preparations were tested for chemotactic and PMN-aggregating activity as described above. RESULTS
Chemotaxigenesis by intact bacteria and
CHEMOTAXIGENESIS BY S. AUREUS CELL WALLS
VOL. 26, 1979
cell surface fractions in normal human serum. Intact bacteria and cell surface fractions, in weights from 0.01 to 1.0 mg, were added individually to 1-ml portions of normal human serum and incubated for 1 h at 370C. Generation of chemotactic activity due to activation of the serum C system (23) by each of these components was expressed as a ratio relative to control serum incubated with PBS. All of the materials tested generated some chemotactic activity at doses as low as 0.1 mg/ml of serum, and, with the exception of intact bacteria, all particles generated significant chemotactic activity at concentrations of 0.01 mg/ml of serum (Fig. 1). None of the cell wall components generated chemotactic activity in serum which had been heat inactivated (560C for 30 min) before incubation with each of the test particles (data not shown). These results indicate that, when compared on an equal-weight basis, CCW, PCW, and PG were approximately equal in their potency as chemotaxigens, and they were more active than intact organisms or cell membranes at doses of 0.1 and 0.01 mg/ml of serum. At higher concentrations (0.5 and 1 mg/ml of serum), however, both intact organisms and cell membranes were capable of significant chemotaxigenesis. The equivalent chemotaxigenesis engendered by the CCW, PCW, and PG preparations would seem to indicate that neither cell wall protein (present in CCW) nor TA (present in both CCW and PCW preparations) plays a significant role in chemotaxigenesis and that PG is the major chemotaxigen. However, since PG makes up 16.0
59
only about half of the cell wall weight (25), there are about two PG particles for every CW particle when these preparations are compared on an equal-weight basis. Therefore, PCW and PG preparations were assessed on both an equalweight and an equal-particle basis. Also, kinetic studies were performed to further evaluate the potencies of these chemotaxigens. These studies were undertaken by incubating 0.1 mg each of intact organisms, CCW, and PCW and 0.1 and 0.05 mg of PG in normal serum for 1, 5, 15, and 60 min and determining the relative rates of generation of chemotactic activity. Figure 2 shows that CCW and PCW elicit a significantly more rapid generation of chemotactic activity than do either PG particles or intact S. aureus H, which at this weight is only minimally chemotaxigenic. Figure 2 also shows that PCW is a more potent chemotaxigen than is PG when compared on a particle-per-particle rather than an equal-weight basis. The faster rate of chemotaxigenesis elicited by CCW and PCW may suggest a role for TA in the generation of chemotactic factors. Given a full hour of incubation time, however, equal weights of PG, CCW, and PCW were essentially equal in the absolute amounts of chemotactic activity generated. Chemotaxigenesis by PCW and PG in other serum sources. To delineate which complement pathway(s) is utilized in the generation of chemotactic activity and to evaluate the role of immunoglobulins in this process, we incusbated PCW and PG particles in normal serum and in MgEGTA-chelated, C2-deficient, and immunoglobulin-deficient sera for 1, 5, 15, and 60 min. As shown in Fig. 3, when these particles were incubated for 60 min in normal serum,
12.0 10.0
10.0
-*
/07aOC organisms
o CCW a
PCW
8.0 *-a PG (l0O/g) o PG (50vg) u-I *_ 6.0 ,
0
- 8.0 O
E
6.0
J
0
E
4.0
>~~~~~~~A -I
4.02.0
2.0
Inuao5 1T 5 mg/mI
0.50.10.01
1 05 00.01
S. aureu H
Crude Cell Walls (CCW)
0.5 0.11 Purified Cell Wolls
1 5 0.1 001 I
Peptidoglycon (PG)
Cell Membranes
(PCW)
FIG. 1. Dose response of chemotaxigenesis. All particles were tested at the indicated weights by incubation in 1 ml of normal serum for 60 min at
370C. Values are averages of three experiments with indicated ranges.
60
Incubation Time (minutes)
I06.50.10.
FIG. 2. Kinetics of chemotaxigenesis elicited by intact organisms and the major cell surface fractions. Intact S. aureus H, CCW, and PCW, each at 100 pg, and PG at 50 and 100 pg were exposed to normal serum for the indicated times, and the chemotactic activity was measured. Values are averages of three
experiments.
60
INFECT. IMMUN.
SCHMELING ET AL.
~~~~~~~~~~~~~~~~def~clent
041
-10.0
2 80 0
E
6.0 4.0
2.0C
5
15
60
5
15
60
Incubation Time (minutes) FIG. 3. Comparison of the kinetics of chemotaxigenesis elicited by PCW(A) or PG (B) in normal, MgEGTAchelated, C2-deficient, or immunoglobulin-deficient serum. A 100-pug amount of either PCW or PG was added to 1 ml of each serum. Chemotaxigenesis was measured after incubation at 37'C for the indicated times. Values represent averages of three experiments.
MgEGTA-chelated serum, and C2-deficient serum, approximately equal amounts of chemotactic activity were generated. When the rates of chemotaxigenesis in these sera were compared, chemotactic activity was generated at a somewhat slower rate in C2-deficient serum than in either normal serum or MgEGTA-chelated serum. Chemotactic activity was generated at a faster rate by PCW than by PG in these sera. The results with C2-deficient serum suggest that chemotaxigenesis can occur in the absence of the classical C pathway, i.e., by alternative pathway activation, although the rate of chemotaxigenesis is decreased in the absence of the classical pathway. When the classical C pathway is blocked by MgEGTA, however, the rate of chemotaxigenesis is relatively unimpaired. The discrepancy between the rate of chemotaxigenesis in the MgEGTA-chelated and C2-deficient sera might possibly be explained by fluid-phase activation of alternative pathway components by Mg2+ (5, 6). Immunoglobulin-deficient serum served as a relatively poor source for chemotaxigenesis by both PCW and PG (Fig. 3). This finding suggests that antibody is necessary for the optimal generation of chemotactic activity by both particles. Effect of solubilization of PCW and PG on chemotaxigenesis in normal human serum. To determine whether enzymatic alteration of particle size would influence the ability of PCW or PG to induce chemotactic activity in
normal serum, we subjected these particles to treatment with the cell wall lytic enzyme lysostaphin for various periods of times. As shown in Fig. 4, this treatment eventually resulted in solubilization of the PCW and PG preparations. The duration of enzymatic treatment of PCW and PG had a marked influence on the chemotactigenic efficiencies of these particles, with a marked reduction in the abilities of these preparations to generate chemotactic activity paralleling the reduction in optical density of the two suspensions. These results indicate that larger particles are more effective than smaller fragments in generating chemotactic activity in serum. In view of the specificity of lysostaphin, the finding that chemotaxigenic activity was abolished upon extensive lysostaphin digestion of PCW and PG indicates that the activities of the intact particles are not due to contaminating material. When isolated TA, a water-soluble cell wall component, was tested for its ability to generate chemotactic activity in normal serum, it was found to be a relatively ineffective chemotaxigen. When 100 1Lg of TA was incubated in normal serum for 60 min, the chemotactic index was 3.6. However, TA was a more potent chemotaxigen than either the PCW or PG preparation that had been solubilized by 2-h lysostaphin treatment. The chemotaxigenic activity of TA may explain the greater potency of PCW as a chemotaxigen compared with PG.
VOL. 26, 1979
CHEMOTAXIGENESIS BY S. AUREUS CELL WALLS
0-
8
-0.42 6.0
(
4.0
E
2.0k
Lysostaphin Treatment (minutes) FIG. 4. Effect of lysostaphin treatment of PCW
and PG on chemotaxigenesis. PCW and PG were subjected to the cell wall lytic enzyme lysostaphin for the indicated times, and these enzymatic digests were incubated in normal serum for 15 min. Dashed lines indicate the optical density of the lysostaphin-treated particles, and solid lines indicate chemotaxigenic activity.
Characterization of the chemotactic factor generated in normal serum. To determine the nature of the chemotactic factor(s) produced in serum by staphylococcal cell wall components, we performed experiments with granulocyte aggregometry. In previous studies,
PMN-aggregating activity in serum or plasma has been attributed to the presence of C5a (3, 11, 17). Normal serum incubated separately with 100 ,tg of intact organisms, PG, or PCW was compared for PMN-aggregating activity with ZAP. All three particles were capable of rendering serum a potent aggregant of normal granulocytes; PCW and PG each generated 90 to 110 ZAP units of activity when incubated in serum, whereas intact organisms yielded 40 to 55 ZAP units in comparable incubations. These results closely paralleled the chemotactic activity exhibited by these sera. When serum activated with 100 jug of PG was subjected to Sephadex G-50 gel filtration, a single peak of chemotactic and PMN-aggregating activity appeared at a molecular weight of about 15,000, similar to the reported molecular weight of C5a (23). Three molecular weight fractions of equal volume were collected: a low-molecularweight fraction (less than 10,000) which yielded a chemotactic index of 0.9, a middle-molecularweight fraction (10,000 to 75,000) which displayed peak PMN-aggregating activity (22 ZAP units) and gave a chemotactic index of 5.9, and a high-molecular-weight fraction (more than 75,000) which displayed no aggregating activity and yielded a chemotactic index of only 0.6 (indices given are the mean values of three separate experiments).
61
Both the chemotactic and PMN-aggregating capacities of activated serum were markedly inhibited by incubation with anti-C5 antiserum, with the chemotactic index of the middle-molecular-weight fraction being reduced from 5.9 to 1.7 and the aggregating activity being reduced from 22 to 3 ZAP units. These findings indicate that C5a is an important chemotactic factor and may be the major chemotactically active fragment generated by incubation of the various cell wall components in human serum.
DISCUSSION Staphylococcal infections are generally characterized by the formation of pus, and this feature suggests the active production of chemotactic factors by the staphylococcus in vivo. Results of recent studies by Russell and colleagues (20) have implicated the serum C system as the chief mediator of neutrophil chemotactic responsiveness to staphylococcal infection. These investigators have concluded that the attraction of neutrophils by factors released from staphylococci and acting directly as chemotaxins is probably trivial. It was the purpose of the current study to evaluate the activities of various staphylococcal cell surface components as chemotaxigens in human serum, i.e., as substances which are capable of generating chemotactic activity in serum.
The results presented demonstrate that all S. aureus H cell surface components tested were capable of functioning as chemotaxigens. On an equal-weight basis, CCW, PCW, and PG were found to be more potent chemotaxigens than were intact organisms or cell membranes. As the intact organisms and the CCW preparation were not significantly more active than the PCW or PG preparations, it would appear that cell wallassociated protein A, which is present in the intact organisms and is anticipated to be present in the CCW but is not found in PCW or PG (25), does not contribute in a major way to the generation of chemotactic activity in normal serum. Other investigators, however, have found that when protein A is isolated from the cell wall, it is capable of activating both the classical and alternative C pathways (19,24) and of generating chemotactic activity (12). It is possible that in the isolated state nonspecific binding of Fc fragments of immunoglobulin to protein A and formation of immune complexes are facilitated and that steric hindrances to these phenomena may occur when protein A is cell wall associated. Miller (16) and Gallin et al. (8) have recently emphasized the importance of studying the kinetics of chemotaxigenesis, as it has been shown that whereas activation of the classical C path-
62
SCHMELING ET AL.
way results in the rapid generation of chemotactic activity, activation of the alternative C pathway is typically characterized by a 15- to 30-min delay before the appearance of chemotactic activity. In the present investigation, kinetic studies revealed that when normal serum was used, CCW and PCW generated chemotactic activity at a more rapid rate than did PG. Also, when PCW and PG were compared on an equal-particle, rather than on an equal-weight, basis as chemotaxigens, PCW was found to be more active than PG. As the major difference between PCW and PG is the absence of TA and its linkage unit in the PG preparation, it appears that when TA is covalently linked to PG the rate of chemotaxigenesis is enhanced. Kinetic studies also revealed that although intact bacteria, PCW, and PG were capable of generating chemotactic activity in the absence of an intact classical C pathway, the rate of chemotaxigenesis was significantly slower in C2deficient serum than in normal serum, suggesting a role for the early components of the classical pathway in this process. An apparent contradiction to this proposal, however, was provided by the finding that the rate of chemotaxigenesis was similar in normal serum and in MgEGTA-chelated serum. Although the explanation for the discrepant findings with C2-deficient serum and MgEGTA-chelated serum has not been established, it is possible that spontaneous fluid-phase activation of alternative pathway components by Mg2e may have artifically enhanced the rate of chemotaxigenesis in MgEGTA-chelated serum, as has been suggested by other investigators (5, 6) using different experimental systems. In serum relatively deficient in immunoglobulins, chemotaxigenesis by the different cell wall preparations developed at a slower rate and to a significantly lesser extent than in normal human serum. These results suggest that immunoglobulins are required for efficient chemotaxigenesis by the staphylococcal cell wall. In support of this conclusion, Grov and Sveen (10) have reported recently that antibodies are necessary for effective chemotaxigenesis by PG in mouse serum.
Experiments with lysostaphin-degraded PCW and PG led to the conclusion that small soluble fragments of the cell wall are relatively inefficient chemotaxigens. Isolated TA, which is water soluble, was found to be a less potent chemotaxigen that were particulate PCW and PG; however, it was more effective than were heavily degraded PCW and PG. These results closely parallel those obtained in C consumption studies recently completed in this laboratory (Wilkinson
INFECT. IMMUN.
et al., manuscript in preparation). That a cell wall particle must be of a certain minimal size for optimal chemotaxigenesis is supported by the work of Grov and Sween (10), who found the critical size of PG fragments to be about 2,000 molecular weight. Snyderman et al. (22) have demonstrated that the major neutrophil chemotactic factor resulting from in vitro C activation is a heat-stable, small-molecular-weight (17,500) cleavage product of the fifth component of complement, designated C5a. Craddock and colleagues (3), as well as O'Flaherty and his co-workers (17), have shown recently that the chemotactic fragment of C5 is capable of inducing PMN aggregation and swelling and that the chemotactic activity of this preparation paralleled its aggregating activity. Gallin and colleagues (9) demonstrated that exposure of granulocytes to preparations of C5a resulted in a decrease in the net negative surface charge of the cell and suggested that a decrease in surface charge is a prerequisite for directed locomotion. Results from the present study, using granulocyte aggregometry as a measure of C5a (3), confirm the importance of this factor in promoting chemotaxis. C5a was found to be present in serum activated by intact bacteria, PCW, and PG. Evidence that C5a is the fragment responsible for chemotactic activity was provided by the observation that anti-C5 antiserum inhibited the ability ofthese activated sera to function as chemoattractants. In conclusion, these in vitro studies demonstrate that several S. aureus cell wall components may be involved in the generation of chemotactic factors in serum. In vivo, these same cell wall components might be expected to contribute as chemotaxigens, leading to the formation of "laudable pus." The optimal generation of the chemotactically active cleavage product, C5a, by these staphylococcal cell wall components appears to depend upon the participation of immunoglobulins and the classical and alternative C pathways. ACKNOWLEDGME14ENTS This work was supported by Public Health Service grants AI 08821, A106931, and HL19725 from the National Institutes of Health and grants-in-aid from the Minnesota Medical Foundation. P.G.Q. is an American Legion Heart Research Professor.
LITERATURE CITED 1. Archibald, A. R. 1972. The chemistry of staphylococcal cell walls, p. 75-109. In J. 0. Bohen (ed.), The staphylococci. Wiley Interscience, New York. 2. Cates, K. L, C. E. Ray, and P. G. Quie. 1978. Modified Boyden chamber methods for measuring polymorphonuclear leukocyte chemotasxis, p. 67-71. In J. I. Gallin (ed.), Leukocyte chemotaxis. Raven Press, New York.
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3. Craddock, P. R., D. E. Hammerschmidt, J. G. White, A. P. Dalmasso, and H. S. Jacob. 1977. Complement (C5a)-induced granulocyte aggregation in vitro: a possible mechansim of complement-mediated leukostasis and leukopenia. J. Clin. Invest. 60:261-264. 4. Craddock, P. R., J. G. White, and H. S. Jacob. 1978. Potentiation of complement (C5a)-induced granulocyte aggregation by cytochalasin-B. J. Lab. Clin. Med. 91: 490-499. 5. Des Prez, R. M., C. S. Bryan, J. Hawiger, and D. G. Colley. 1975. Function of the classical and alternative pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgCl2-ethylene glycol tetraacetic acid. Infect. Immun. 11:1235-1243. 6. Fine, D. P. 1977. Comparison of ethyleneglycoltetraacetic acid and its magnesium salt as reagents for studying alternative complement pathway function. Infect. Immun. 16:124-128. 7. Forsgren, A., R. H. McLean, A. F. Michael, and P. G. Quie. 1975. Studies of the alternate pathway in chelated serum. J. Lab. Clin. Med. 85:904-912. 8. 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 pathway. Clin. Immunol. Immunopathol. 3:334-346. 9. Gallin, J. I., J. R. Durocher, and A. P. Kaplan. 1975. Interaction of chemotactic factors with the cell surface. I. Chemotactic factor induced changes in human granulocyte surface charge. J. Clin. Invest. 55:967-974. 10. Grov, A., and K. Sveen. 1978. Induction of leukochemotaxis by peptidoglycan of Staphylococcus aureus. Acta Pathol. Microbiol. Scand. Sect. B 86:375-378. 11. Hammerschmidt, D. E., P. R. Craddock, J. McCullough, R. S. Kronenberg, A. P. Dalmasso, and H. S. Jacob. 1978. Complement activation and pulmonary leukostasis during nylon fiber filtration leukopheresis. Blood 51:721-730. 12. Harvey, R. L, G. Kronvall, G. M. Troup, R. F. Anderson, and R. C. Williams, Jr. 1970. Chemotaxis of polymorphonuclear leukocytes by protein A of the staphylococcus. Proc. Soc. Exp. Biol. Med. 135:453456. 13. Heymer, B., and E. T. Rietschel. 1977. Biological properties of peptidoglycans, p. 344-349. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 14. Keller, H. V., M. W. Hess, and H. Cottier. 1974. The in
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vitro assessment of leukocyte chemotaxis. Antibiot. Chemother. (Washington, D.C.) 19:112-125. 15. Kim, Y., P. S. Friend, L G. Dresner, E. G. Yunis, and A. F. Michael. 1977. Inherited deficiency of the second component of complement (C2) with membranoproliferative glomerulinephritis. Am. J. Med. 62:765-771. 16. Miller, M. E. 1975. Pathology of chemotaxis and random mobility. Semin. Hematol. 12:59-82. 17. O'Flaherty, J. T., D. L. Kreutzer, and P. A. Ward. 1978. Chemotactic factor influences on the aggregation, swelling, and foreign surface adhesiveness of human leukocytes. Am. J. Pathol. 90:537-550. 18. Peterson, P. K., B. J. Wilkinson, Y. Kim, D. Schmeling, S. D. Douglas, P. G. Quie, and J. Verhoef. 1978. The key role of peptidoglycan in the opsonization of Staphylococcus aureus. J. Clin. Invest. 61:597-609. 19. Pryjma, J., K. Pryima, A. Grov, and P. B. Heczko. 1976. Immunological activity of staphylococcal cell wall antigens, p. 873-881. In J. Jeljasweicz (ed.), Staphylococci and staphylococcal disease. Gustav Fisher Verlag, New York. 20. Russell, R. J., P. C. Wilkinson, R. J. Mchnroy, S. McKay, A. C. McCartney, and J. P. Arbuthnott. 1976. Effects of staphylococcal products on locomotion and chemotaxis of human blood neutrophils and monocytes. J. Med. Microbiol. 8:433-449. 21. Schindler, C. A., and V. T. Schuhardt. 1964. Lysostaphin: a new bacteriolytic agent for the staphylococcus. Proc. Natl. Acad. Sci. U.S.A. 51:414-421. 22. Snyderman, R., H. S. Shin, J. K. Phillips, H. Gewurz, and S. E. Mergenhagen. 1969. Polymorphonuclear leukocyte chemotactic activity in rabbit serum and guinea pig serum treated with immune complexes: evidence for C5a as the major chemotactic factor. Infect. Immun. 1:521-525. 23. Snyderman, R., H. S. Shin, J. K. Phillips, H. Gewurz, and S. E. Mergenhagen. 1969. A neutrophil chemotactic factor derived from C'5 upon interaction of guinea pig serum with endotoxin. J. Immunol. 103:413-422. 24. Stilenheim, G., 0. Gotz, 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. 25. Wilkinson, B. J., Y. Kim, P. K. Peterson, P. G. Quie, and A. F. Michael. 1978. Activation of complement by cell surface components of Staphylococcus aureus. Infect. Immun. 20:388-392.
