Fig. 4

The pore-forming toxins RTX and VasX do not intoxicate A. castellanii. Distribution of V. cholerae wild-type (WT) and a rtxA-deficient mutant strain (a) or WT and a strain with an impaired type VI secretion system (T6SS) (ΔvipA; deficient for VasX secretion) (b) within infected A. castellanii at 20 h p.p.c. The considered compartments were the CV of trophozoites, the CV of cysts, and the cytosol of cysts before and after lysis. Values represent averages from three independent experiments (±s.d., as shown by the error bars). c, d The type VI secretion system of toxigenic strain ATCC25872 is constitutively on. c V. cholerae strains A1552 (O1 El Tor; pandemic) and ATCC25872 (O37; non-pandemic) carrying a gene that encodes a translational fusion between the T6SS sheath protein VipA and sfGFP (vipA-sfgfp) at the native locus of vipA were grown for 3.5 h in LB. The images depict cells visualized in the phase contrast (Ph), in the green (GFP) channel, and an overlay of both channels. Scale bar: 5 μm. d The constitutive T6SS activity of strain ATCC25872 was confirmed in an interspecies killing assay in which E. coli served as prey. Prey survival was assessed by counts of colony-forming units (CFUs) after plating on selective medium. Values represent average from three independent experiments (±s.d.). e Hemolysin-based hemolytic activity might mask effects of the T6SS in ATCC25872. V. cholerae A1552 and ATCC25872 as well as their ΔhlyA, ΔvipA, and ΔhlyAΔvipA variants were tested for hemolytic activity on blood agar plates. f Mutant V. cholerae A1552 and ATCC25872 strains lacking hemolysin (ΔhlyA) or hemolysin and the T6SS core protein VipA (ΔhlyAΔvipA) were assessed for their intra-amoebal localization at 20 h p.p.c. as described for a. Values are averages from three independent biological experiments