Figure 4. Sip-Sop synergy revealed by cis BENEFIT screening.

A. Schematic illustrating cis BENEFIT screening. Pair-wise combinations of effector-augmented (d-effector) S.typhimurium strains [enhanced effector shown in capitals, e.g. aceBp (dSopB) and aCebp (dSipC) produce increased levels of SopB and SipC, respectively] were mixed 50∶50 (overall MOI 50), where one strain additionally carries spectinomycin resistance (Sp^R). The mixed inoculum is used to infect cultured cells, and a reciprocal infection performed in parallel in which the opposing strain is spectinomycin resistant. After infection (60 min), extracellular bacteria are killed with gentamicin and infected cell lysate dilutions replica plated on LB agar (depicted white) and LB containing spectinomycin (grey). Overall invasion rate and invasion of the spectinomycin resistant strain are calculated by scoring colony-forming units. The invasion rate of the strain lacking the marker is calculated by subtracting invasion of the spectinomycin resistant strain from the overall value (X₁, Y₂). To correct for the mild influence of spectinomycin on invasion efficiency, invasion rates of individual strains are averaged with the parallel experiment i.e. [X₁+X₂ ^Sp]÷2 = X and [Y₁+Y₂ ^Sp]÷2 = Y. Each pair of infections were performed 4 times in triplicate. B. S.typhimurium SL1344 or effector-augmented (d-effector) strains were mixed pair wise (50∶50; MOI 50) for infection. Invasion of each strain was assessed using selectable markers after 60 min (Figure 4A). Results are the mean of four independent experiments each performed in triplicate Upper: Pie charts depict total invasion by each combination (size; combined %) and relative contribution of each strain (division; %). Lower: Tables show difference (%) in total invasion rate (left) and relative invasion rate of each strain (right) after correction. Shading denotes a significant increase (green) or no significant change (grey) in invasion rate (Mann Whitney U p<0.05).