Vol. 20, No. 3
INFECTION AND IMMUNITY, June 1978, p. 770-775 0019-9567/78/0020-0770$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Dichotomy Between Opsonization and Serum Complement Activation by Encapsulated Staphylococci PHILLIP K. PETERSON,'* YOUNGKI KIM,2 BRIAN J. WILKINSON,"3 DAVID SCHMELING,2 ALFRED F. MICHAEL,2 AND PAUL G. QUIE2 Departments of Medicine,' Pediatrics,2 and Microbiology,:' University of Minnesota School of Medicine, Minneapolis, Minnesota 55455
Received for publication 29 December 1977
Previous studies have demonstrated that encapsulated Staphylococcus aureus strains are not effectively opsonized by the serum complement system. Encapsulated staphylococci thereby "resist phagocytosis." To test whether this phenomenon might be explained by an inability of encapsulated strains to activate complement, the relationship between staphylococcal opsonization and serum complement activation was studied. Although encapsulation was found to interfere with opsonization by pooled human serum (human polymorphonuclear leukocytes phagocytized significantly fewer encapsulated bacteria than unencapsulated bacteria after incubation in this opsonic source), encapsulated (S. aureus M and Smith diffuse) and unencapsulated (S. aureus M variant and Smith compact) strains had similar capacities for complement activation as measured by C3-C9 consumption. When C2-deficient and immunoglobulin-deficient sera were studied, again C3-C9 consumption was not influenced by the presence or absence of a capsule. In addition, C3 was detected on the surface of both S. aureus M and M variant strains after incubation in pooled serum and staining with fluorescein-conjugated anti-C3 antibody. Thus, encapsulated staphylococci are not effectively opsonized even though complement is activated and C3 is present on the bacterial surface. The exact mechanism by which the capsule interferes with opsonization is still not known; however, inhibition of complement activation appears not to be the explanation of this phenomenon. Previous investigators have shown that encapsulated Staphylococcus aureus strains are able to "resist phagocytosis" (8, 12). Although the mechanism for this interference with phagocytosis is not clearly established, it has been suggested that this phenomenon is related to the special opsonic requirements of encapsulated staphylococci (12, 17). Results of recent studies indicate that encapsulated strains are not effectively opsonized by either heat-labile serum factors (the classical and alternative pathways of the complement system) or heat-stable opsonic factors present in nonimmune human serum (15). The present study was undertaken to examine the hypothesis that encapsulation interferes with opsonization by preventing complement activation at the bacterial cell surface, thereby limiting formation of the ligand C3b and attachment of bacteria to phagocytic cells. MATERIALS AND METHODS Bacterial strains and radioactive labeling. S. aureus M (encapsulated), M variant (unencapsulated), Smith diffuse (encapsulated), and Smith compact (unencapsulated) strains were kindly provided by
M. A. Melly, Vanderbilt University School of Medicine, Nashville, Tenn. Descriptions and classification of three of these strains have been presented elsewhere (12, 18, 25). The S. aureus M variant strain produces nonmucoid colonies and does not possess a visible capsule; however, it has some of the properties of the encapsulated M strain (clumping factor negative, diffuse colonies in serum soft agar [M. A. Melly, personal communication]). All strains were maintained on nutrient agar plates at 40C. Radioactive labeling was achieved by inoculating several colonies into 20 ml of peptone-yeast extract broth (6) containing 0.04 mCi of [2-3H]glycine (specific activity, 5 to 15 Ci/mmol; New England Nuclear, Boston, Mass.). After 18 h of incubation at 370C, the bacteria were washed three times in phosphate-buffered saline (PBS), pH 7.4, and resuspended in PBS to the desired final concentration (109, 4 x 109, or 10"' colony-forming units [CFU]/ml) using a spectrophotometric method confirmed by pour-plate colony counts. Serum sources. Sera collected from four healthy donors were pooled. Serum was obtained from a donor with a genetically determined, complete, selective deficiency of C2 (7) and from a 62-year-old male with common variable immunodeficiency (immunoglobulin G [IgGI, 73 mg; IgM, 7 mg; and IgA,
INFECTION AND IMMUNITY, June 1978, p. 770-775 0019-9567/78/0020-0770$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Dichotomy Between Opsonization and Serum Complement Activation by Encapsulated Staphylococci PHILLIP K. PETERSON,'* YOUNGKI KIM,2 BRIAN J. WILKINSON,"3 DAVID SCHMELING,2 ALFRED F. MICHAEL,2 AND PAUL G. QUIE2 Departments of Medicine,' Pediatrics,2 and Microbiology,:' University of Minnesota School of Medicine, Minneapolis, Minnesota 55455
Received for publication 29 December 1977
Previous studies have demonstrated that encapsulated Staphylococcus aureus strains are not effectively opsonized by the serum complement system. Encapsulated staphylococci thereby "resist phagocytosis." To test whether this phenomenon might be explained by an inability of encapsulated strains to activate complement, the relationship between staphylococcal opsonization and serum complement activation was studied. Although encapsulation was found to interfere with opsonization by pooled human serum (human polymorphonuclear leukocytes phagocytized significantly fewer encapsulated bacteria than unencapsulated bacteria after incubation in this opsonic source), encapsulated (S. aureus M and Smith diffuse) and unencapsulated (S. aureus M variant and Smith compact) strains had similar capacities for complement activation as measured by C3-C9 consumption. When C2-deficient and immunoglobulin-deficient sera were studied, again C3-C9 consumption was not influenced by the presence or absence of a capsule. In addition, C3 was detected on the surface of both S. aureus M and M variant strains after incubation in pooled serum and staining with fluorescein-conjugated anti-C3 antibody. Thus, encapsulated staphylococci are not effectively opsonized even though complement is activated and C3 is present on the bacterial surface. The exact mechanism by which the capsule interferes with opsonization is still not known; however, inhibition of complement activation appears not to be the explanation of this phenomenon. Previous investigators have shown that encapsulated Staphylococcus aureus strains are able to "resist phagocytosis" (8, 12). Although the mechanism for this interference with phagocytosis is not clearly established, it has been suggested that this phenomenon is related to the special opsonic requirements of encapsulated staphylococci (12, 17). Results of recent studies indicate that encapsulated strains are not effectively opsonized by either heat-labile serum factors (the classical and alternative pathways of the complement system) or heat-stable opsonic factors present in nonimmune human serum (15). The present study was undertaken to examine the hypothesis that encapsulation interferes with opsonization by preventing complement activation at the bacterial cell surface, thereby limiting formation of the ligand C3b and attachment of bacteria to phagocytic cells. MATERIALS AND METHODS Bacterial strains and radioactive labeling. S. aureus M (encapsulated), M variant (unencapsulated), Smith diffuse (encapsulated), and Smith compact (unencapsulated) strains were kindly provided by
M. A. Melly, Vanderbilt University School of Medicine, Nashville, Tenn. Descriptions and classification of three of these strains have been presented elsewhere (12, 18, 25). The S. aureus M variant strain produces nonmucoid colonies and does not possess a visible capsule; however, it has some of the properties of the encapsulated M strain (clumping factor negative, diffuse colonies in serum soft agar [M. A. Melly, personal communication]). All strains were maintained on nutrient agar plates at 40C. Radioactive labeling was achieved by inoculating several colonies into 20 ml of peptone-yeast extract broth (6) containing 0.04 mCi of [2-3H]glycine (specific activity, 5 to 15 Ci/mmol; New England Nuclear, Boston, Mass.). After 18 h of incubation at 370C, the bacteria were washed three times in phosphate-buffered saline (PBS), pH 7.4, and resuspended in PBS to the desired final concentration (109, 4 x 109, or 10"' colony-forming units [CFU]/ml) using a spectrophotometric method confirmed by pour-plate colony counts. Serum sources. Sera collected from four healthy donors were pooled. Serum was obtained from a donor with a genetically determined, complete, selective deficiency of C2 (7) and from a 62-year-old male with common variable immunodeficiency (immunoglobulin G [IgGI, 73 mg; IgM, 7 mg; and IgA,
