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Supplementary methods

Fig. S1. Purification of ZmpC from TIGR4 culture supernatant.

Culture supernatant of TIGR4 was concentrated 200x with a 100 kDa cut-off amicon device and loaded on a Superdex 75 gel filtration column.

A. Collected fractions (0.5 ml) were separated by SDS-PAGE and the gel was silverstained.

B. Neutrophils were treated with the gel filtration fractions (1–12) for 30 min at 4°C. Cells were stained with PE-labelled anti-PSGL-1 (KPL1) for 30 min at 4°C. The percentage PSGL-1 staining was determined by comparison of mean fluorescence with untreated cells. The proteins of interest were identified by mass-spectrometry as: ZmpB, ZmpC and Serine protease of Streptococcus pneumoniae.

C. Purity of ZmpC after large-scale purification from TIGR4ΔZmpB culture supernatant analysed by SDS-PAGE and Instant-blue staining.

D. The effect of 10% serum on PSGL-1 cleavage induced by ZmpC. PSGL-1 on human neutrophils was stained with PE-conjugated mAb KPL1 and measured by flow cytometry. The percentage PSGL-1 staining was determined by comparison of mean fluorescence with untreated cells. Data represent the mean ± s.e.m. of three independent experiments.

E. Anti-ZmpC IgG detected in the sera of 12 healthy volunteers.

F. Neutrophils in pulmonary interstitium measured by MPO activity in lung homogenates 8 h after onset of disease.

Table S1. Strains used in this study.

Table S2. Primers used in this study.

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