Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum
Fig 9
Endogenous ecotin protects E. coli against C3 and C5b-9-deposition from NHS.
The extent and the initiating pathway of CS activation were studied on two wild type and ecotin KO coli strains, the polymannan LPS carrying ATCC 23505 and the smooth LPS containing ATCC 12014. Cells were treated with increasing concentrations of NHS and labeled for deposited C3 fragments, and C5b-9 as described in Materials and methods. Mean fluorescence intensity (MFI) values of C3 positive cells (A, D) and percentage of C5b-9/PI positive cells inside the bacteria FSC-SSC gate were determined and is shown (C, F). Cells were treated with 2% NHS containing the indicated inhibitors, and the MFI of deposited C3 was determined as for panel A and D. MFI of deposited C3 without inhibitor was considered 100% complement activity (B, E). Data represent median +/- SD of three independent experiments. MFI of the C3-positive and C5b-9/PI positive ATCC 23505 ecotin KO cells was orders of magnitude higher than that of the wild type cells (A, C). This strain should trigger the LP, and indeed, SGMI-1 and SGMI-2, inhibiting MASP-1 and MASP-2, respectively, as well as exogenously added ecotin provided complete inhibition, while the anti-C1q mAb had no effect. The broad-specificity protease-inhibitor, FUT-175 was included as a control (B). Similar trends were found with the ATCC 12014 cells. This strain is less intensely attacked by the complement, and while it is also protected by its endogenous ecotin, the ratio of C3 MFI values and percentage of C5b-9/PI positive cells are smaller (D, F). All exogenously added inhibitors decreased C3-deposition on ATCC 12014 ecotin KO cells, but SGMI-1 and anti-C1q mAb were the most efficient. Therefore, this strain was attacked by all three, but dominantly by the classical and the alternative pathway (E).