Figure 1. ECP but not I13A is able to agglutinate bacteria.

(A) E. coli, P. aeruginosa and A. baumanii cells were incubated with 5 µM of ECP or I13A mutant in microscopy plates during 4 h and stained with syto9 (live cells, green) and propidium iodide (dead cells, red). Images were taken at 0, 0.5 and 3 h using a Leica SP2 confocal microscopy as described in the Materials and Methods section. Scale bar represents 50 µm. Arrows were depicted to show cell agglutination. Images depicted are representative from two independent experiments. (B) E. coli cells were incubated with 5 µM of ECP or I13A mutant during 4 h and samples were analyzed using a FACSCalibur cytometer. FSC-H is the low-angle forward scattering, which is roughly proportional to the diameter of the cell and SSC-H is the orthogonal or side scattering, which is proportional to cell granularity or complexity. Agglutination is registered as an increase in both scattering measures. In all experiments cell cultures were grown at exponential phase (OD₆₀₀ = 0.2) and incubated with proteins in 10 mM sodium phosphate buffer, 100 mM NaCl, pH 7.4. The plots are representative of three independent experiments.