Vol. 21, No. 1

INFECTION AND IMMUNITY, July 1978, p. 7-9 0019-9567/78/0021-0007$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Lung Clearance of Staphylococcus aureus Strains with Differing Protein A Content: Protein A Effect on In Vivo Clearance GARY N. GROSS,* STANLEY R. REHM, GALEN B. TOEWS, DAVID A. HART,

AND

ALAN K.

PIERCE

Pauline and Adolph Weinberger Laboratory for Cardiopulmonary Research, Department of Internal Medicine, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 Received for publication 14 December 1977

We have studied the clearance from murine lungs of two strains of Staphylococcus aureus, one possessing high and the other possessing low levels of protein A. S. aureus FDA 209 and S. aureus Wood 46 were assayed for their ability to bind mouse immunoglobulin G, using an indirect radioimmunoassay. S. aureus FDA 209 binding of mouse immunoglobulin was significantly greater than that of S. aureus Wood 46 (118,909 versus 37,845 cpm). Clearance of these two strains from the lung after a 30-min aerosol inoculation period was not significantly different. The percentage of bacteria remaining in the lung was 49.2 and 55.0% at

2 h, 31.8 and 33.2% at 3 h, and 25.4 and 17.2% at 4 h for protein A-rich and protein A-poor strains, respectively (P > 0.20 at each time). These data suggest that the previously demonstrated in vitro antiphagocytic effect of protein A may not be relevant to pulmonary clearance mechanisms. bacteria. The IgG fraction of goat anti-mouse immunoglobulin (Cappel Laboratories, Cochranville, Pa.) was labeled with NalnI by a modified lactoperoxidase technique (1). The Formalin-killed bacterial suspension (0.5 ml) was incubated with 10 1d of normal mouse serum for 30 min at room temperature in glass tubes (12 by 75 mm). After incubation, each sample was washed three times at 230 x g for 8 min in PBS with a bovine serum albumin carrier (1 mg/ml). After the final wash, the bacterial pellet was resuspended in 0.5 ml of PBS, 10 1I of '25I-labeled goat anti-mouse immunoglobulin was added, and the tube was shaken. In preliminary experiments we have shown that this amount of goat antisera does not show differential binding to Wood 46 or FDA 209 until normal mouse serum is added. This mixture was incubated for 30 min at room temperature and washed three times in PBS. The final suspension of bacteria was collected over a 0.45-nm filter above a vacuum apparatus. The filters were then counted in an automatic gamma counter. Controls of sera without bacteria and bacteria without sera were included. Each sample was run in triplicate, and the average number of counts per minute minus background (bacteria without serum) was used for analysis. Female BALB/c mice weighing 18 to 20 g were exposed to the bacterial aerosol in a Henderson aerosol apparatus, using a previously described chamber for 66 mice (10). After 30 min of exposure, animals were removed and divided into four approximately equal groups. Animals were sacrificed immediately (zero time) and at 2, 3, and 4 h after aerosolization by subluxation of the spine and cross-clamping of the trachea. By using aseptic techniques, the lungs were removed, placed in 4 ml of distilled water in a sterile

Protein A is a cell wall constituent of most strains of Staphylococcus aureus (7) which has the capacity to bind the Fc portion of immunoglobulin G (IgG) (3). Protein A-IgG complexes thus formed have been shown to activate the complement system (11), and it has been suggested that protein A may activate complement in the absence of inmmunoglobulin (9). These properties of protein A have been thought responsible for the antiphagocytic activity demonstrated by in vitro studies (2, 5). The present study was undertaken to examine this issue in vivo by comparing lung clearances of a protein A-rich and a protein A-poor strain of S. aureus. MATERIALS AND METHODS S. aureus FDA 209 and Wood 46 were kindly provided by Eugene Rosenblum. These organisms were maintained on Trypticase soy agar slants, and 18-h cultures were grown in Trypticase soy broth for aerosolization. Bacteria were washed twice in 0.9% sterile saline for 15 min at 2,600 x g, resuspended in sterile saline to a standard optical density at 650 nm, and placed in a Collison nebulizer. Bacteria used for the IgG-binding assay were taken from the Collison nebulizer after the aerosol period and suspended in 0.3% Formalin in phosphate-buffered saline (PBS), pH 7.2, and remained at room temperature for 24 h. These Formalin-killed organisms were washed twice and resuspended in PBS to a final concentration of approximately 108 organisms per ml. An indirect radioimmunoassay was used to determine relative amounts of immunoglobulin bound to 7

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INFECT. IMMUN.

GROSS ET AL.

glass flask, and ground with a Virtis 45 tissue homogenizer and Broeck tissue grinder to insure complete rupture of all phagocytes. A 0.1-nil portion of the resultant homogenate was then diluted in 0.9 ml of distilled water, and serial 10fold dilutions were plated on Trypticase soy agar. Colony-forming units were counted after overnight incubation at 370C. An index of clearance (R value) was obtained by determining the percentage of original bacteria deposited which remained in the lung at each time interval for each animal as follows: R = (number of bacteria in lung at time t/mean number of bacteria in lungs of similarly aerosolized animals at zero time) x 100.

TABLE 2. Percentage of staphylococci remaining R value

Strain

Time (h)

Wood 46

0 2 3 4

(±SEMa~) 100

55.0 (±6.6) 33.2 (±3.9) 17.2 (±2.7)

0 100 2 49.2 (±3.3) 3 31.8 (±1.5) 4 25.4 (±3.2) a SEM, Standard error of the mean.

FDA 209

No. of animals

15 16 16 15

15 16 16 15

RESULTS A significant difference (P < 0.001) was demonstrated in the quantity of immunoglobulin bound by FDA 209 compared with Wood 46. Results from three normal littermates of the aerosolized animals (sera 1, 2, and 3) are shown in Table 1. This difference was anticipated from previous studies demonstrating that the former strain possesses higher levels of protein A than does the latter (9) and confirms the presence of this binding ability in the organisms aerosolized in these experiments. The differing conditions of the aerosol system on the 2 exposure days resulted in an initial (zero time) deposition of 1.4 (±0.11) X 10" for FDA 209 and 9.4 (±1.5) x 105 for Wood 46 strains of S. aureus. The rate of clearance of staphylococci has been shown to be unaffected by this range of inoculum (8). There was no significant difference in the clearance of the two strains of S. aureus at any of the time intervals examined (Table 2). The percentages of bacteria remaining in the lungs (R values) of protein A-rich and -poor strains, respectively, were 55 and 49% at 2 h, 33 and 32% at 3 h, and 17 and 25% at 4 h (P > 0.2 at each interval by Mann-Whitney U test).

DISCUSSION The findings in this study indicate that the content of protein A in S. aureus strains is not critical to their rates of clearance from normal TABLE 1. IgG binding to S. aureus cpma

1

2 3

126,914 109,834 119,979

ACKNOWLEDGMENTS This investigation was supported by a Regent's Appropriation Grant and by Public Health Service grant HL-21827 from the National Heart, Lung, and Blood Institute. S.R.R. is a Postdoctoral Research Fellow sponsored by the American Lung Association, Texas, and G.B.T. is a Postdoctoral Fellow of the Parker B. Francis Foundation. LITERATURE CITED

Serum no. FDA 209

lungs. These findings differ from previous in vitro experiments assessing the effect of protein A on phagocytosis (2, 5, 9) and provide data regarding in vivo mechanisms, a subject only alluded to previously (5). The differences in in vitro and in vivo data may be explained by a number of factors. Complement activation by protein A-IgG complexes is thought to be responsible for the antiphagocytic effect of protein A in in vitro systems (2, 5), whereas in vivo data indicate that complement is not required for optimal clearance of S. aureus from the lungs (unpublished observation). Also, the in vitro experiments have been performed with polymorphonuclear leukocytes as the primary phagocyte (2, 5), whereas in vivo experiments suggest that the alveolar macrophage is the principal phagocyte involved in anti-staphylococcal defense of the lungs (6). These studies suggest that protein A does not play a role in the infectivity of S. aureus in the lungs of healthy animals. The difference in in vitro and in vivo findings further indicates the importance of studying certain aspects of bacterial defense in the intact animal and in the organ system in question.

Wood 46

39,441 35,584 38,509

Mean (±SEMb) 118,909 (+4,959) 37,845 (+1,162) None 21,445 35,521 a Counts of "nI-labeled anti-mouse immunoglobulin per minute. b SEM, Standard error of the mean. P < 0.001.

1. Baur, S., E. S. Vitetta, C. J. Sherr, L. Schenkein, and J. W. Uhr. 1971. Isolation of heavy and light chains of immunoglobulin from the surfaces of lymphoid cells. J.

Immunol. 106:1133-1135. 2. Dossett, J. H., G. Kronvall, R. C. Williams, Jr., and P. G. Quie. 1969. Antiphagocytic effects of staphylococcal protein A. J. Immunol. 103:1405-1410. 3. Forsgren, A. 1968. Protein A from Staphylococcus aureus. VI. Reaction with subunits from guinea pig y,and y2-globulin. J. Immunol. 100:927-930. 4. Forsgren, A., and K. Nordstrom. 1974. Protein A from Staphylococcus aureus: the biological significance of its

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LUNG CLEARANCE OF S. AUREUS STRAINS

reaction with IgG. Ann. N.Y. Acad. Sci. 236:252-266. 5. Forsgren, A., and P. G. Quie. 1974. Effects of staphylococcal protein A on heat labile opsonins. J. Immunol. 112:1177-1180. 6. Goldstein, E., W. Lippert, and D. Warshauer. 1974.

Pulmonary alveolar macrophage. Defender against bacterial infection of the lung. J. Clin. Invest. 54:519-528. 7. Jensen, K. 1958. A normally occurring Staphylococcus antibody in human serum. Acta Pathol. Microbiol. Scand. 44:421-428. 8. Laureuzi, G. A., L. Berman, K First, and E. H. Kass. 1964. A quantitative study of the deposition and clearance of bacteria in the murine lung. J. Clin. Invest.

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43:759-768. 9. Peterson, P. K., J. Verhoef, L D. Sabath, and P. G. Quie. 1977. Effect of protein A on staphylococcal opsonization. Infect. Immun. 15:760-764. 10. Southern, P. M., Jr., A. K. Pierce, and J. P. Sanford. 1968. Exposure chamber for 66 mice suitable for use with the Henderson aerosol apparatus. Appl. Microbiol. 16:540-542.

11. Stilenheim, G.,

0. Gotze, N. R. Cooper, J. Sjoquist, and H. J. Mijler-Eberhard. 1973. Consumption of human complement components by complexes of IgG with protein A of Staphylococcus aureus. Immunochemistry 10:501-507.

Lung clearance of Staphylococcus aureus strains with differing protein A content: protein A effect on in vivo clearance.

Vol. 21, No. 1 INFECTION AND IMMUNITY, July 1978, p. 7-9 0019-9567/78/0021-0007$02.00/0 Copyright i 1978 American Society for Microbiology Printed i...
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