Figure 5. Perforin-2 forms pores in bacterial surfaces after infection and in Perforin-2 overexpressed eukaryotic membranes that are visible by negative stain transmission electron microscopy (TEM).

(A, B) Electron micrograph of polymerized Perforin-2 membrane lesions from Perforin-2-GFP transfected HEK-293 cells, with Perforin-2 activated to form pores by trypsin digestion to the enriched membrane fraction. Panel A Demonstrates the quantity of pores on the Perforin-2 overexpressed membranes after trypsin activation. Panel B denotes a higher magnification to illustrate the uniform pore structure. (C-G) Perforin-2 wild-type MEFs were treated with IFN-γ for 14 hr, and infected with (C–E) MRSA or (F, G) M. smegmatis. After 5 hr the infected bacteria were isolated and imaged utilizing negative stain TEM. Arrows point to black, stain-filled pores on the bacterial cell wall surrounded by white, stain excluding borders created by polymerized Perforin-2. Round pores measure 8.5–10 nm inner diameter, the size typical for polymerized Perforin-2-pores. Panels E and G are close-up images of the boxed region in C and F. Blinded quantification of pore amount with different conditions is demonstrated in Figure 6—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.06508.020

Figure 5—figure supplement 1. Quantification of pores from negative stain transmission electron microscopy.

10 blinded fields were counted from the above eight conditions. Pores are defined as regions containing stain (corresponding to a ‘hole” in the surface) that is approximately 10 nm in diameter. Around the “hole” ultrastructure needs to be visible to ensure that the presumed “hole” is actually a pore. Statistical analysis was performed by with one-way ANOVA with Tukey Post-hoc Multiple Comparisons.*p < 0.05.

DOI: http://dx.doi.org/10.7554/eLife.06508.021