Complement is a critical element of the innate immune system, and in vitro studies support the importance of the complement system in the opsonophagocytic killing of both CP++, CP+, and CP− S. aureus
). Verbrugh et al. (26
) showed that the CP++ strain M incubated with NHS had C3 localized on the cell wall, beneath the capsular layer. The thick capsular layer was antiphagocytic because it interfered with the recognition of cell wall-bound C3b and iC3b molecules by the phagocytic cell receptors.
Little attention has been given to the details of the molecular interactions between S. aureus
and complement proteins. Studies on the interaction between complement components and the clinically relevant serotype 5 and 8 strains of S. aureus
have not been reported. One study examined the effects of complement depletion on staphylococcus-induced lethality in mice (7
). Easmon and Glynn (7
) compared the virulence of two CP++ strains, a CP+ strain (type 8), and two CP− strains in normal or complement-depleted mice that were challenged intraperitoneally. Their results indicated the C3 depletion increased mouse lethality following challenge with all of the strains except for one CP− strain. The results of our experiments expand the findings of Easmon and Glynn by challenging mice by the i.v. route with a clinically relevant S. aureus
CP+ type 5 isolate. Only 8% of normal animals died from infection with CP+ strain Reynolds, whereas 64% of the complement-depleted animals succumbed to the infection.
Although the classical and alternative pathways both contribute to C3 deposition on CP+ S. aureus strains, we showed that the relative contribution of each pathway was dependent on complement protein concentrations. At a high serum concentration (20%) that may reflect that in blood, the alternative pathway was very active and accounted for 90% of the total C3 binding to S. aureus cells. At a lower serum concentration (2%) that might be present in tissues, the classical complement pathway predominated. Under the latter conditions with NHS, the classical pathway activated C3 binding to S. aureus, even in the absence of added specific antibody. IgG greatly enhanced C3 binding to a CP+ staphylococcal strain in hypogammaglobulinemic serum, confirming the importance of antibody in the classical pathway.
The results of C3 deposition experiments comparing the CP+ strain with its isogenic CP− mutant were dependent on the culture conditions under which the staphylococci were grown. C3 binding to S. aureus
was evaluated under different bacterial growth conditions to reflect different disease processes. Mid-logarithmic-phase staphylococcal cultures may emulate organisms growing in the bloodstream, whereas organisms growing on solid medium may more closely mimic organisms growing on a surface, like a damaged heart valve or an intravascular catheter. For broth-grown S. aureus
harvested in the mid-logarithmic phase of growth, the amounts of C3 bound to the CP+ and CP− isogenic pairs of organisms were identical. This is consistent with the observation that organisms in the logarithmic phase of growth do not produce detectable levels of CP5 (22
; this study). Like other agr
-regulated staphylococcal virulence factors, S. aureus
CP is maximally expressed in post-exponential-phase cultures (22
). When the CP+ strain Reynolds was cultivated under conditions of maximal capsule production (stationary-phase broth cultures or on agar plates), C3 deposition was reduced by ~90%. Thus, an organism that may be adequately opsonized in bloodstream log-phase growth may not be adequately opsonized on a heart valve or catheter when in stationary phase. However, C3 deposition on the CP− mutant JL022 was also diminished markedly when the strain was grown to stationary phase or on solid medium. Thus, there are bacterial factors in addition to capsule that decrease C3 binding to stationary-phase staphylococci. The nature of these growth phase-dependent factors is unknown at the present time. In a comparison of bacterial strains grown on solid media, CP++ strains bound 50% fewer C3 molecules than did CP+ strains, and CP+ strains bound 42% fewer C3 molecules than CP− strains. These results suggest that C3 deposition also diminishes as capsule thickness increases.
The C3 binding data for S. aureus
cultivated on solid medium are consistent with results obtained with capsule type 7 Streptococcus pneumoniae,
in which increased capsule production correlated with decreased C3 binding (4
). Previously reported studies, in which S. aureus
was cultivated on solid media, revealed that the CP+ strain Reynolds showed increased lethality for mice in vivo compared to a CP− mutant strain (25
). Similarly, strain Reynolds sustained a higher level of bacteremia in infected mice and was cleared from the bloodstream less readily than a CP− mutant strain (25
). CP+ S. aureus
strains have been shown to be susceptible to phagocytic killing only in the presence of specific capsular antibodies and complement, whereas CP− S. aureus
strains were opsonized for phagocytosis by nonimmune serum with complement activity (16
Studies by Gordon et al. (12
) with S. aureus
strain ATCC 25923 found significant amounts of C3b and iC3b bound to the surface as well as moderate amounts of C3d. We found that C3 fragments bound to the surface of CP+ S. aureus
strains are a mixture of C3b and iC3b but did not find any C3d. There are receptors for these two forms of C3 on leukocytes, and both promote opsonophagocytosis (1
). A large proportion of the bound C3 fragments was shed from the surface of both CP+ and CP− organisms, predominantly in the iC3b form.
The ease of cleaving iC3b on the organisms with minute concentrations of trypsin suggests that iC3b on the surface of S. aureus is as exquisitely sensitive to cleavage as it is on the surface of particles such as sheep red blood cells. We speculate that in an abscess containing proteolytic enzymes released from dying neutrophils, the iC3b may be cleaved and shed from the organism surface to an even greater extent, further decreasing the susceptibility of S. aureus to phagocytosis.
In summary, we show in this report that complement is important in host immune defense against acute staphylococcal infection provoked by a clinically relevant CP+ S. aureus isolate. Both C3b and iC3b are deposited in approximately equal amounts on the surface of CP+ and CP− staphylococci. CP+ strains partially inhibit complement-mediated opsonization by diminished C3 binding and subsequent shedding of the C3 fragments from the bacterial surface. With the use of isogenic CP+ and CP− mutants, we show that decreased C3 binding is capsule dependent, whereas C3 shedding is not. Stationary-phase organisms had <10% binding of C3 and may be poorly opsonized and thus poorly phagocytosed in NHS. Further studies will examine whether these mechanisms are an important means of host defense evasion for S. aureus.