Fig. 2: Tse5 membrane insertion and pore-formation requires cleaved Tse5-CT.
a The top panel shows a phospholipid monolayer assembly in a typical Langmuir–Blodgett balance experiment. A monolayer is formed at the air-buffer interface, and the protein is introduced into the buffer. Changes in the monolayer’s lateral pressure are recorded over time until reaching the equilibrium (middle panel). The ability of Tse5 (black), the Tse5-ΔCT (green), Tse5-K47G-P48A (blue) and Tse5-D1141A (red) variants to insert into the hydrophobic core of lipid monolayers spontaneously is calculated by extrapolating the fitted curve to ΔΠ = 0. The critical lateral pressure (Πc) values for Tse5, Tse5-K47G-P48A, Tse5-D1141A, and Tse5-ΔCT are 34.74 mN m−1, 34.58 mN m−1, 29.05 mN m−1, and 27.17 mN m−1, respectively. The dotted line indicates the threshold value of lateral pressure consistent with unstressed biological membranes. Each dot corresponds to an independent experiment, representing the lateral pressure increase (ΔΠ) as a function of initial lateral pressure (Π0; n = 10 for each protein). b The top panel shows a schematic representation of a planar lipid bilayer assembly using the modified solvent-free Montal-Mueller technique. Two chambers (cis/trans) are separated by a lipid bilayer, and a 250/50 mM gradient is generated. Protein is added on the cis side. Representative current traces were obtained before (Control) and after the addition of Tse5 or the variants Tse5-K47G-P48A, Tse5-D1141A and Tse5-ΔCT. All tested variants display current spikes indicative of an interaction between the protein and the lipid bilayer, while stable channels are observed for Tse5 and Tse5-K47G-P48A but not with Tse5-D1141A or Tse5-ΔCT. In all traces, dashed lines indicate zero current levels. The applied voltage was 100 mV for the current spikes, while in the IV curves, it is indicated by grey numbers. The recordings were digitally filtered at 500 Hz using a low-pass 8-pole Bessel filter for better visualisation. The lower panel shows the permeability ratios for the Tse5 C-terminal fragment (Tse5-CT)29, Tse5 and the Tse5-K47G-P48A variant. The borders of the boxes define the 25th and 75th percentiles, and the line within each box marks the median. Whiskers indicate the standard deviation of the mean. Data are means of 7 (Tse5-CT, grey), 12 (Tse5, black), and 5 (Tse5-K47G-P48A, blue) independent experiments. Solid circles correspond to the individual data points with the minima and maxima being 4.06 and 7.41 for Tse5-CT, 2.63 and 5.56 for Tse5, and 1.75 and 7.10 for Tse5-K47G-P48A c In vivo relevance of the Tse5 propeptide cleavage. Bacterial competition assays demonstrate that the cleavage of the Tse5-NT is not essential, while the cleavage of the Tse5-CT is essential for toxicity. Data are represented for the surviving Tse5-susceptible P. aeruginosa PAO1 prey strain following 20 h of competition with donor P. aeruginosa PAO1 strains with an active T6SS and either expressing the wild-type Tse5 (Parent) or variants Tse5-K47G-P48A (K47G-P48A), Tse5-D1141A (D1141A) or not expressing Tse5 (Δtse5). Each bacterial competition was done in triplicate (n = 3). The mean with standard deviation (SD) and the value of each replicate are plotted. Statistical significance was evaluated with the ordinary one-way ANOVA with Dunnett’s multiple comparison test. P-values of Parent vs K47GP48A, D1141A, and Δtse5 are 0.9898, 0.0006, and 0.0052, respectively. P-value > 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****). Plotting and data analysis were done with GraphPad Prism v9.5.
