Fig 1. The native cell wall inhibits Ply activity and release.

(A) Washed whole cells, subcellular fractions (cell wall and protoplast), or sonicated cell lysates of wildtype pneumococcus were tested for hemolytic activity as described in Materials and Methods. Protoplasts were lysed by sonication prior to the experiment. The hemolytic activity of each sample is represented as a percent of the total as determined for the sonicated cell lysate. (B) To determine if the activity of intact cells is due to cell-associated or secreted Ply, washed whole cells were incubated with SRBCs or buffer alone for one hour. Cells incubated with buffer alone were pelleted and the cell-free supernatant was removed and tested for hemolytic activity as described in Materials and Methods. Of note, Ply is the only active hemolysin under these conditions; deletion of ply completely abolishes hemolytic activity. Columns represent the mean and error bars denote SEM of at least four (A) or two (B) biological replicates.