Fig. 4.

The hlyU mutation and its complement did not significantly affect the cytotoxicity of V. anguillarum against ASK cells. (A) ASK cells were incubated with various strains of V. anguillarum at an MOI of 100 for 4 h at 20°C. The data showed that strain S305 (hlyU mutant) still caused ASK cells to round-up, as did wild-type strain M93Sm and strain S307 (hlyU complement). ASK cells treated with NSS buffer (mock) exhibited no rounding or detachment during the course of the experiment. The photograph magnification is 100×. (B) LDH release from ASK cells treated with wild-type M93Sm (•), hlyU strain S305 (▵), hlyU complement S307 (▴), and rtxA vah1 double mutant S183 (○) at various MOI (20, 50, 100, and 200) for 4 h. LDH release was measured in relative fluorescence units (RFU) and then calculated to yield the percentage of cytotoxicity according to the instructions of the manufacturer. The incubation time at 0 was the base level of LDH in ASK cells treated with NSS buffer. Data are the representative of three separate experiments done with three replicates. Error bars show the standard deviation of the average. *, statistical difference of strain S305 from the wild-type treated data (P > 0.1).