Figure 2

The S1P-specific aerolysin/ETX pore-forming domain-containing protein CC1G_11805 is toxic for C. elegans and A. aegypti . (A) Multiple sequence alignment of the aerolysin/ETX pore-forming domain-containing proteins C. perfringens epsilon toxin (Cper_etx), CC1G_11805, CC1G_10308 and CC1G_08369. (B) Expression of soluble CC1G_11805 in soluble form in E. coli BL21. Whole cell protein extracts in PBS were produced from non-induced (WCE) and induced (WCE + IPTG) E. coli BL21/pET24-CC1G_11805 cultures. Supernatants from consecutive low (LS) and high (HS) speed centrifugations were collected and run on a 12% SDS-PAGE to evaluate CC1G_11805 expression and solubility. CC1G_11805-mediated toxicity for A. aegypti (C) and C. elegans (D) larvae was assessed as described previously [40]. E. coli BL21 expressing the previously characterized fungal lectin CGL2 was used as positive control for toxicity against A. aegypti Rockefeller. IPTG-induced E. coli bearing an empty pET24 vector (EV) was used as a negative control. Columns represent the mean percentage of surviving insect larvae (C), or worms either dying or reaching the indicated developmental stage (D) from 4 replicates each. SDs are shown as error bars. Dunn’s multiple comparisons were used to test the statistical significance of the toxicity observed in the A. aegypti assay. Mann Whitney test was used to compare the percentage of worms reaching each developmental stage when treated with EV or CC1G_11805. *: 0.01 < p-value < 0.05; **: 0.001 < p-value < 0.01.