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. 2022 Nov 3;14(1):2138677. doi: 10.1080/19490976.2022.2138677

Figure 5.

Figure 5.

B. thetaiotaomicron-derived indole inhibits the enhancement of EPEC T3SS activity upon co-culture with V. cholerae. (a) Supernatants of WT B. thetaiotaomicron (B. theta) grown for 2, 4, 6 and 8 h and ΔtnaA B. thetaiotaomicron grown for 8 h were added to co-cultures of EPEC and V. cholerae grown in DMEM: BHI mixture aerobically, for 6 h. The bacterial pellets and supernatants (bacterial sup) were separated, normalized, and analyzed. The secreted proteins were concentrated from culture supernatants and analyzed via 12% SDS-PAGE and western blotting using an anti-EspB antibody. The expression of the effector protein Tir, was analyzed by subjecting the bacterial pellets to SDS-PAGE and western blotting using an anti-Tir antibody. Samples were also probed with anti-DnaK to confirm equal loading. (b) A schematic overview of the bacterial combinations and indole supplementation used for this experiment. WT EPEC was sub-cultured in a mixture of 1:1 (v/v) DMEM: BHI medium as pure culture (sample 1), a co-culture with V. cholerae (sample 2), a co-cultured with B. thetaiotaomicron (sample 6), or a tri-culture with V. cholerae and B. thetaiotaomicron (sample 4 with WT B. thetaiotaomicron and sample 5 with ΔtnaA B. thetaiotaomicron). B. thetaiotaomicron were pre-grown for 8 h before EPEC and V. cholerae were added. One of the EPEC and V. cholerae co-cultures was supplemented with 500 µM indole (sample 3). Pure cultures of WT and ΔtnaA B. thetaiotaomicron were used as negative controls (samples 7 and 8). All cultures were grown anaerobically for 6 h and their indole concentrations were determined (average values are highlighted in yellow). The bacterial pellets and supernatants (bacterial sup) were separated, normalized, and analyzed as described in panel A. (c) Relative light production was used to assess the levels of CAI-1 produced by WT V. cholerae when grown alone or in co-culture with either B. thetaiotaomicron (B. theta) or EPEC. Synthetic CAI-1 (10 µM) and the supernatant of ΔcqsA V. cholerae strain, which cannot produce CAI-1, were used as positive and negative controls, respectively. Data are averaged from three replicates of a representative experiment.