Capsule production and growth phase influence binding of complement to Staphylococcus aureus

Abstract

Complement-mediated opsonization of bacteria by C3 binding is an important component of the host innate immune system. Little information is available concerning the interaction between complement proteins and capsule type 5 and 8 Staphylococcus aureus strains, even though these isolates are responsible for approximately 70% of human staphylococcal infections. To investigate the importance of an intact complement pathway in an experimental staphylococcal infection, control and C3-depleted mice were challenged intravenously with 10(7) CFU of a serotype 5 S. aureus isolate. Whereas only 8% of the control mice succumbed to the infection, 64% of the complemented-depleted animals died. In vitro parameters of C3 binding to two heavily encapsulated (CP++) strains, three encapsulated (CP+) strains, and an isogenic capsule-negative (CP-) mutant were examined. The alternative pathway contributed 90% of C3 binding in 20% serum at 30 min, whereas it accounted for only 13% of C3 binding in 2% serum. Stationary-phase organisms bound only 10% as much C3 as mid-log-phase organisms; this was only in part due to capsule. When the S. aureus strains were cultivated on solid medium, the CP++ isolates bound 50% less C3 than CP+ strains; a CP+ strain bound 42% less C3 than the CP- mutant. Both C3b and iC3b fragments of C3 bound to S. aureus cells, and about one-third of the bound C3 was shed from the staphylococcal surface as iC3b, regardless of the CP phenotype of the strain. Thus, the phase of growth and presence of capsule are critical to opsonization.

FIG. 1

Kaplan-Meier plot illustrating increased lethality of CP+ S. aureus Reynolds in mice after C3 depletion by injection with CVF compared with normal mice. Two experiments were performed, with a total of 14 CVF-treated mice and 13 mice not receiving CVF. All mice received 10⁷ CFU of S. aureus Reynolds (CP5). The lethality for CVF-treated mice was 64%, compared with a lethality of 8% for mice not receiving CVF (P = 0.004).

FIG. 2

C3 deposition onto mid-logarithmic-phase CP+ strains Lowenstein (serotype 5) and Wright (serotype 8) as a function of increasing serum concentration. Deposited C3 molecules were detected using radiolabeled goat anti-human C3 antibody. Maximal C3 deposition on the organism was achieved in ≥10% serum. Each data point represents the mean of four independent experiments with single determinations. Error bars represent SEM.

FIG. 3

C3 deposition on mid-log-phase CP+ strain Lowenstein over time in 20% serum (A) and 2% serum (B) for all complement pathways (GVBS⁺⁺ buffer) and the alternative pathway only (Mg-EGTA-GVBS buffer). Deposited C3 molecules were detected using radiolabeled goat anti-human C3 antibody. In 20% serum the alternative pathway accounted for >90% of total C3 deposition by 30 min. In 2% serum the alternative pathway deposited C3 on the organisms relatively slowly compared to C3 deposition by all pathways. Each data point represents the mean of four independent experiments with single determinations. Error bars represent SEM.

FIG. 4

C3 deposition on mid-logarithmic-phase CP+ strain Reynolds for hypogammaglobulinemic serum and hypogammaglobulinemic serum + IVIg. Trace ¹²⁵I-C3 was added to serum to detect C3 binding. One set of bacterial samples was coated with antibody by preincubation in IVIg (1 mg/ml) prior to incubation in hypogammaglobulinemic serum. In 2% hypogammaglobulinemic serum about two-thirds less C3 was deposited than in normal serum, but much of the C3 deposition was restored by precoating the bacteria with antibody contained in IVIg. The data are from a representative experiment. Error bars indicate standard deviations.

FIG. 5

C3 fragments deposited on CP+ strain Reynolds and CP− mutant JL022 grown in liquid media to mid-logarithmic phase or stationary phase or grown on solid medium. Trace ¹²⁵I-C3 was added to 10% plasma to detect C3 binding. At least six separate determinations were made for each value. Error bars represent SEM.

FIG. 6

C3 deposition onto CP+ strain Lowenstein, CP+ strain Reynolds, CP++ strain Smith, and CP++ strain M grown to the mid-logarithmic-phase of growth (A) and grown on solid-media (B). Trace ¹²⁵I-C3 was added to 10% serum to detect C3 binding. When grown to mid-log-phase, average C3 binding to CP+ and CP++ organisms is not significantly different (P >0.05), except between strains Smith and Lowenstein. When grown on solid media, average C3 binding to CP++ organisms is significantly less than C3 binding to CP+ organisms (P <0.001). At least three separate determinations were made for each value. Error bars represent SEM.

FIG. 7

Deposited C3 fragments on EA or CP+ strain Reynolds that were unaffected or released by treatment with 25 mM methylamine. The samples were analyzed by SDS-PAGE and Western blotting with anti-C3 antibodies. The 104-kDa band represents the C3b α′ chain, the 75-kDa band represents the C3 β chain, the 63-kDa band represents iC3b α′₁, and the 42-kDa band represents iC3b α′₂. Whereas the C3 fragments bound to EA are almost all from iC3b, both C3b and iC3b forms are found bound to strain Reynolds.

FIG. 8

Total C3 shed (A) and C3 fragments shed (B) from opsonized CP+ strain Reynolds or CP− mutant JL022 cultivated in liquid medium to mid-log phase or on solid medium. ¹²⁵I-C3-coated organisms were incubated in neutral buffer for 60 min at 30°C. Total C3 shed averaged ∼32% regardless of the CP phenotype of the S. aureus strain or the growth conditions. At least three separate determinations of each value were made. Error bars represent SEM. SDS-PAGE and autoradiography were used to detect C3 fragments shed from opsonized strain Reynolds. The 104-kDa band represents the C3b α′ chain, the 75-kDa band represents the C3 β chain, the 63-kDa band represents iC3b α′₁, and the 42-kDa band represents iC3b α′₂. The majority of the shed C3 fragments are from iC3b.

FIG. 9

Deposited iC3b fragments released by incubating ¹²⁵I-C3-coated CP+ strain Reynolds or CP− mutant JL022 (cultivated on solid medium) with trypsin (2 μg/ml) or control buffer (HBSS) for 10 min at 30°C. About 40% of deposited C3 fragments were in the iC3b form, as detected by their susceptibility to treatment with trypsin. Error bars represent SEM.