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. 2019 Sep 13;10:2128. doi: 10.3389/fmicb.2019.02128

Figure 2.

Figure 2

Immediate activation of the T3SS can be measured by an in vitro β-lactamase assay. (A) Accumulative effector secretion into the culture supernatant during a standard in vitro secretion assay using Y. enterocolitica MRS40. At the time points indicated (0 min = activation of T3SS secretion by resuspension of bacteria in secreting medium, see also time line at bottom), the culture supernatant of 3 × 109 bacteria was removed and visualized by Coomassie staining of an SDS-PAGE gel. Control (far right), bacteria resuspended in non-secreting medium. (B) Quantification of effector export in the indicated time ranges after resuspension of Y. enterocolitica ΔHOPEMTasd in secreting medium (see also time line at bottom). Red bars, β-lactamase activity indicative of export of the reporter T3SS substrate YopH1−17-β-lactamase; green bars, non-secreting control; gray bars, β-lactamase lacking a T3SS secretion signal under secreting conditions. Error bars indicate standard deviation of the averages of technical triplicates between three biological replicates. *p < 0.05 vs. the YopH1−17-β-lactamase, switch to secreting conditions, sample (red bars) in a two-tailed homoscedastic t-test. Secreting and non-secreting (non-secr.) conditions refer to incubation in medium with addition of 5 mM EGTA or CaCl2, respectively.

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