Figure 2.

Hemolytic activity and quantification of pneumolysin (PLY) produced by wild-type, ply-deleted, and ply-recoded Streptococcus pneumoniae serotype 3 strains. A, Hemolytic activity of PLY in the supernatant of growing S. pneumoniae strains measured over time. *P < .003 (1-way analysis of variance). All strains had similar growth kinetics. B, Concentration of PLY in growth media of the indicated strains 7 h after initiation of the culture, calculated from hemolytic units as described elsewhere [22]. *P < .001 comparing A66.1 and A66:PM4; *P < .002 comparing A66.1 and A66:PM2; **P < .004 comparing A66:PM2 and A66:PM4; ^#P < .001 comparing WU2 and WU2:PM4 (Student unpaired t test of 4–5 independent experiments). C, Total amount of PLY produced in vitro as measured by enzyme-linked immunosorbent assay from stationary phase cultures. *P < .01 comparing A66.1 and A66:PM4; *P < .04 comparing A66.1 and A66.1:PM2; **P = .051 comparing A66:PM2 and A66:PM4 (Student unpaired t test. D, Western blot of total PLY produced after in vitro growth reached the stationary phase. Lane 1, A66.1; lane 2, A66:PM2; lane 3, A66:PM4; lane 4, A66:Δply; lane 5, purified PLY (500 ng). All bands indicate similar molecular weights (52 kDa). OD₄₀₅, optical density at 405 nm.