FIG 5.

E. tarda T3SS and FliC are required for cytotoxicity for fish leukocytes. (A) Fish leukocytes isolated from head kidney were left noninfected or were infected at an MOI of 5 with different strains. After 1 h and 2 h, the levels of LDH released were analyzed. Values are means ± SD for 8 replicate cultures. ***, P < 0.001. (B) The release of LDH by fish leukocytes is inhibited by Ac-YVAD-AOM. Primary leukocytes were pretreated with 317 μM Ac-YVAD-AOM or DMSO for 90 min and then infected with the wild type or the esaN mutant for 2 h. Culture supernatants were collected to determine LDH release. ***, P < 0.001. (C) FliC2HA is secreted into the culture supernatant in a T3SS-dependent manner. Bacterial strains carrying pACYC-fliC2HA were grown in TSB for preparing protein samples from bacterial pellets (P) or culture supernatants (S). Membranes were probed with anti-HA for FliC2HA, anti-DnaK (a bacterial cytosolic marker), and anti-EvpC (a T6SS protein, which served as an internal loading control for different strains). DnaK was not detected in any culture supernatants, showing that detection of FliC2HA is not due to contamination from bacterial cells.