StcE reduces the classical complement-mediated erythrocyte lysis by human serum. 1 μl human serum was untreated or treated with increasing concentrations of StcE′-His or BSA overnight before the addition of 10⁷ opsonized sheep erythrocytes for 1 h at 37°C, in triplicate. Unlysed cells were pelleted and the OD₄₁₂ of the supernatant was measured in a spectrophotometer (*, P = 0.0077; **, P = 0.0028; unpaired t test). Suspension in distilled water was used to determine the absorbance of 100% lysis of erythrocytes.

StcE binds erythrocyte surfaces. (A) 250 ng StcE′-His or Alexa-StcE′-His was incubated with opsonized sheep erythrocytes for 10 min at 37°C, after which the cells were washed and the binding of StcE was detected in a flow cytometer. (B) Increasing concentrations of Alexa-StcE′-His (0.1–25.6 μg) were added to sheep erythrocytes. StcE binding was detected by flow cytometry as described above, and the geometric mean fluorescence for each sample was plotted against the corresponding concentration of Alexa-StcE′His.

Cleavage of C1-INH by StcE is not necessary to bind erythrocytes or provide protection against classical complement. (A) Classical complement-mediated erythrocyte lysis was determined as described in Materials and Methods in the presence of 8 μg C1-INH, C1-INH and 1 μg StcE′-His, or C1-INH and an enzymatic point mutant of StcE, 1 μg StcE′ E435D-His (*, P < 0.005; unpaired t test). (B) 2 μg C1-INH was untreated or treated with 1 μg StcE′-His or StcE′ E435D-His before the addition of sheep erythrocytes as described in Materials and Methods. Erythrocytes were washed and polyclonal goat anti–human C1-INH IgG was added to cells. C1-INH binding was detected with FITC-conjugated anti–goat IgG in a flow cytometer.

StcE interacts with the aminoterminal domain of C1-INH. (A) StcE-treated C1-INH is nonreactive with mAbs against the NH₂ terminus of and RCL-inserted C1-INH. 1 μg virgin C1-INH was untreated or treated with 1 μg StcE′-His or 2 μg kallikrein overnight at room temperature, separated by electrophoresis on 8% reducing SDS-PAGE gels, transferred to nitrocellulose, and analyzed with a polyclonal anti–human C1-INH Ab (left), mAb 3C7 (middle), or mAb 4C3 (right). (B) StcE does not cleave C-serp(98), a recombinant C1-INH molecule truncated at amino acid 98. COS-7 cells were transfected with hC1-INH/pcDNA3.1(−) or C-serp(98)/pcDNA3.1(−) and metabolically labeled with [³⁵S]methionine. 100 μl supernatants were untreated or treated with 10 μg StcE′-His overnight, immunoprecipitated with polyclonal anti–human C1-INH IgG protein A–Sepharose, and separated by electrophoresis on a 10% reducing SDS-PAGE gel.

Model of the mechanism by which StcE potentiates C1-INH-mediated inhibition of classical complement. (1) StcE interacts with the cell surface. (2) StcE binds the NH₂-terminal domain of C1-INH, sequestering the serpin to the cell surface. Alternatively, StcE may interact with C1-INH before binding to the cell surface. (3) Cell-bound C1-INH binds to C1r and/or C1s of the C1 complex via the RCL, inactivating the serine protease. (4) StcE cleaves within the NH₂ terminus of C1-INH, releasing the serpin/serine protease complex and a smaller, aminoterminal cleavage fragment of C1-INH from the cell surface. (5) StcE binds another C1-INH molecule and repeats the cycle as described above. Note that the figure is not drawn to scale.

StcE potentiates C1-INH–mediated inhibition of classical complement. (A) 16 μg C1-INH was untreated or treated with increasing concentrations of StcE′-His or BSA overnight at room temperature before the addition of 1 μl human serum and 10⁷ opsonized sheep erythrocytes, in triplicate. Erythrocytes were incubated at 37°C for 1 h, pelleted, and the OD₄₁₂ of the supernatant was measured in a spectrophotometer. The point indicated by • lacked C1-INH. (B) Increasing amounts of C1-INH were untreated or treated with 1 μg StcE′-His overnight at room temperature before the addition of human serum and opsonized sheep erythrocytes as described in A, in triplicate. Suspension in distilled water was used to determine the absorbance of 100% lysis of erythrocytes (all p-values <0.0005; unpaired t test).

C1-INH binds erythrocyte surfaces in the presence of StcE. (A) 8 μg C1-INH was untreated or treated with 1 μg StcE′-His overnight at room temperature. C5-deficient human serum was added to opsonized sheep erythrocytes in the presence of 8 μg C1-INH, 1 μg StcE′-His, C1-INH, and StcE′-His, or buffer alone (mock) for 10 min at 37°C. Erythrocytes were washed and polyclonal goat anti–human C1-INH IgG was added to cells. C1-INH binding was detected with FITC-conjugated anti–goat IgG in a flow cytometer. (B) 8 μg C1-INH was incubated with or without 1 μg StcE′-His overnight at room temperature before the addition of 50 mM imidazole and Ni-NTA agarose beads to remove StcE′-His from the sample. Opsonized sheep erythrocytes were added for 10 min at 37°C and C1-INH binding was detected as described. As a control for StcE-treated C1-INH binding, StcE′-His was not removed from one sample before analysis. (C) Increasing concentrations of C1-INH (0.25–16 μg) were mixed with 1 μg StcE′ E435D-His overnight at room temperature before the addition of opsonized erythrocytes. C1-INH binding was detected in a flow cytometer as described above, and the geometric mean fluorescence for each sample was plotted against the corresponding concentration of C1-INH. (D) 5 μg C1-INH was untreated or treated with 5 μg StcE′-His or StcE′ E435D-His for 10 min before being immunoprecipitated with an anti–C1-INH Ab. Precipitated proteins were separated by reducing SDS-PAGE, transferred to nitrocellulose, and probed with an anti-StcE Ab as described in Materials and Methods.

StcE is unable to potentiate C1-INH in the absence of cells. (A) Increasing concentrations of C1-INH (0–1.0 μg) were mixed with 250 ng StcE′-His overnight at room temperature and incubated with 100 ng kallikrein for 1 h at 37°C before the addition of S-2302, in triplicate. Absorbance of the chromogenic substrate was measured at 410 nm in a spectrophotometer after 30 min at room temperature. 100% kallikrein activity was determined in the absence of C1-INH. (B) An excess of purified, activated C1s (1.5 μg) was untreated or treated with 100 ng C1-INH in the absence or presence of 50 ng StcE′-His or StcE′ E435D-His for 1 h at 37°C before being separated by nonreducing SDS-PAGE and analyzed by immunoblot with an anti-C1s Ab. The gray arrow indicates the presence of uncomplexed C1s and the black arrow indicates the presence of C1s bound to C1-INH in an SDS-insoluble complex.

StcE provides increased serum resistance to E. coli K-12 by the potentiation of C1-INH. 8 μg C1-INH was untreated or treated with 1 μg StcE′-His overnight. E. coli strain C600 was grown in LB broth at 37°C to midlogarithmic phase and diluted at 1:10 into a buffer containing 2% human serum and either 8 μg C1-INH, 1 μg StcE′-His, C1-INH, and StcE′-His, or no additional protein, in triplicate. Bacteria were incubated at 37°C for 1 h, serially diluted, and plated onto LB agar. Percent survival was determined by comparing the number of CFUs to bacteria incubated under the same conditions without serum (*, P = 0.0037; unpaired t test).