E. coli K1 did not exhibit proteolytic activities in zymography. (A) Neuropathogenic E. coli K1 strain E44 and E. coli K-12 laboratory strain HB101 were grown overnight with shaking under aerobic condition at 37°C in RPMI 1640. Next day the optical density was adjusted to 0.22 for K1 and 0.35 for K-12 using 595 nm wavelength yielding approximately 1 × 108 per mL bacterial c.f.u. Various bacterial counts were mixed with lysis buffer and loaded onto 7.5% SDS-PAGE gel containing gelatin as substrate, while A. castellanii (104 cells) was used as a positive control. After electrophoresis, gels were washed for four times with 2.5% Triton X-100 solution for 30 min and incubated overnight in developing buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 5 mM CaCl2, 500 μM MgCl2 and 2 μM ZnCl2). Following this incubation, gels were stained with Coomassie Brilliant Blue, destained and finally visualized. (B and C) E. coli K1 and K12 were grown overnight with shaking under aerobic condition at 37°C in RPMI 1640 with or without 10% serum fetal calf serum. Next day, cells were removed by centrifugation and cell free conditioned mediums were collected and 10 μl of these were loaded along with uninoculated medium on 7.5% gelatin (B) and collagen (C) substrates zymography gels as described in A. Note that neither whole bacteria nor their conditioned medium degraded gelatin/collagen in zymographic assays tested. The results are representative of three independent experiments.