B.G.J.S designed and performed research and wrote the article; K.T., S.R.J., I.S.H., M.M.V and A.v.d.E. performed the research; K.T., E.A.M.S., J.A.G.v.S., and C.J.C.d.H. participated in designing research; and all authors checked the final version of the manuscript. The authors declare no competing financial interests.
Pneumococcal immune evasion: ZmpC inhibits neutrophil influx
Article first published online: 3 MAY 2013
© 2013 John Wiley & Sons Ltd
Volume 15, Issue 10, pages 1753–1765, October 2013
How to Cite
Surewaard, B. G. J., Trzciński, K., Jacobino, S. R., Hansen, I. S., Vughs, M. M., Sanders, E. A. M., van der Ende, A., van Strijp, J. A. G. and de Haas, C. J. C. (2013), Pneumococcal immune evasion: ZmpC inhibits neutrophil influx. Cellular Microbiology, 15: 1753–1765. doi: 10.1111/cmi.12147
- Issue published online: 15 SEP 2013
- Article first published online: 3 MAY 2013
- Accepted manuscript online: 18 APR 2013 06:26AM EST
- Manuscript Accepted: 4 APR 2013
- Manuscript Revised: 21 MAR 2013
- Manuscript Received: 30 NOV 2012
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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