Fig 1.
Modeling predicts that victim lysis rate determines T6SS killing efficiency.
(A, C) Simulation snapshots compare attacker versus susceptible strain competition outcomes (see legend) for T6SS firing rates 50 and 150 firings cell-1 h-1 and for slow and rapid victim lysis. (B, D) Magnified sections of simulated communities showing occupation of interstrain boundary by lysing cells for the higher firing rate case shown in (A, C). (E, F) Snapshots of 3D competition simulations (“sim.”) after 13 hours’ growth, for slow (E) and rapid (F) victim lysis (kfire = 100.0 firings cell-1 h-1). (G) Parameter sweep measuring relative fitness of the T6+ attacker strain (ωT6+ / ωT6−, equivalent to ratio of strain division rates) for increasing attacker firing rate; solid lines denote means. (H) Comparison of normalized (“Norm.”) peak killing rates from simulations (circles) with those predicted from the fraction of the interstrain boundary occupied by lysing cells (the “boundary saturation”; dashed lines, see Materials and methods). Parameter values: Nhits = 1, c = 0.001; “slow” and “rapid” victim lysis correspond respectively to klysis = 0.8, 8.0 h-1 throughout. Five simulation replicates are shown for each parameter combination; additional parameter values and quantification of boundary saturation are shown in S1 Fig and S2 Fig. Raw data are available at dx.doi.org/10.6084/m9.figshare.11980491. T6SS, type VI secretion system.
Fig 2.
Attackers with lytic T6SS effectors outperform nonlytic analogs in microfluidic and agarose competition assays.
Comparison of T6SS competition dynamics in microfluidic chambers, varying T6SS effector type: T6SS+ attacker A. baylyi with lytic effector Tae1 (A, C, E) or nonlytic effector Tse2 (B, D, F). (A, B) Fluorescent microscopy images (“Dead stain”) show unlabeled E. coli victim cells between 2 groups of T6SS+ A. baylyi (green) within observation channels. Propidium iodide dead stain (magenta) labels DNA released from lysed cells (Tae1, A), or DNA inside cells upon membrane permeabilization (Tse2, B). (C, D) Fluorescent microscopy time-lapse series (“Experiment,” left column) show dynamics of competition (A. baylyi, green; E. coli, magenta); images are representative of 16 biological replicates. Simulations of chamber competitions using the agent-based model from Fig 1 are shown alongside (“Simulation,” right column; attacker, susceptible, and lysing cells shown in green, magenta, and white, respectively). (E, F) Each strain’s channel occupancy was measured in 5-minute intervals based on fluorescence signal and plotted (E. coli in magenta, A. baylyi in green) as percentage of the whole chamber (solid lines, “exp.”). Data correspond to a single representative replicate; additional replicates are shown in Fig 3E and S3 Movie. These data are shown alongside analogous plots for chamber simulations (dashed lines, “sim”; lines and patches, respectively, denote means and standard deviations of 5 simulation replicates). Scale bars: 2 μm. (G, H) Competition assays carried out on agarose plates for same attacker and victim strains as in A–F, along with parental and T6SS-knockout (Δhcp) controls. Percentage area occupancies of each strain (G) were computed from fluorescence micrographs (S5 Fig). Two-way ANOVA (α = 0.01) with Tukey post hoc test; **** p ≤ 0.0001; n = 18 spot competitions analyzed per group. (H) Cell recovery data for these assays quantify A. baylyi and E. coli survival for the same 4 treatment groups, subsample of 3 replicates per case. Raw data are available at dx.doi.org/10.6084/m9.figshare.11980491. CFU, colony-forming unit; exp., experiment; sim., simulation; hcp, hemolysin-coregulated protein; T6SS, type VI secretion system; Tae1, type VI amidase effector 1; Tse2, type VI effector 2.
Fig 3.
Osmoprotective conditions prevent victim cell lysis and killing without affecting toxin activity.
(A) Time-lapse images show T6SS firing and killing dynamics between cells grown on agarose pads in presence and absence of osmoprotectant (OP+, OP–, respectively). Propidium iodide (“Dead stain” magenta) labels DNA released from susceptible E. coli (unlabeled) upon T6SS attack from A. baylyi cells (vipA-sfGFP, green) armed with toxin Tae1. Arrowheads mark formation of spheroplasts upon T6SS intoxication (PI does not label intoxicated cells under the OP+ condition). (B) Hcp was precipitated from culture supernatants of A. baylyi with and without OP, with proteins separated by SDS-PAGE and stained using Coomassie Blue. Column M contains protein reference ladder; axis values show approximate protein molecular weight in kDa. (C) Image analysis of time lapses comparing E. coli death rates, normalized by E. coli–A. baylyi initial contact counts, in the presence and absence of OP (2-group t test; p-value = 0.9027; confidence interval for difference in means −0.288<μOP+−μOP−<0.256). Data originate from ten 45-minute time lapses as in (A), also shown in S6 Movie. Boxplot shows data ranges (dashed lines), interquartile ranges (blue boxes), and means (red lines). (D) Fluorescence time-lapse series of microfluidic competition assay between A. baylyi armed with Tae1 and E. coli in the presence of OP at indicated time points (hh:min). Images are representative for 16 biological replicates. (E) Quantification of chamber dynamics plot initial (t0), maximum (tmax), and final (tend) area occupancies of E. coli (magenta) and A. baylyi (green) from microfluidic competition assays. Data for indicated A. baylyi strains and treatments are displayed. Two-way ANOVA (α = 0.01) with Tukey post hoc test; ****p ≤ 0.0001; n = 16 channel competitions were analyzed per group. (F) A. baylyi and E. coli recovery data for agarose competition experiments are shown in presence (+) or absence (–) of OP. For each OP+ condition, we show an additional treatment with 20 mM EDTA added to ensure lysis of spheroplasts pre-recovery. Scale bar: 2 μm. Raw data are available at dx.doi.org/10.6084/m9.figshare.11980491. CFU, colony-forming unit; exp., experiment; Hcp, hemolysin-coregulated protein; ns, not significant; OP, osmoprotectant; sfGFP, super-folding green fluorescent protein; T6SS, type VI secretion system; Tae1, type VI amidase effector 1; Tse2, type VI effector 2; VipA, ClpV-interacting protein A.
Fig 4.
Taxonomy showing distribution of lytic and nonlytic T6SS toxins across the Proteobacteria.
Each species is positioned in the tree according to its taxonomic classification in the NCBI taxonomy database and colored according to Proteobacterial Class (α, β, δ, ε, γ). Colored circles indicate the T6SS effectors associated with a single, randomly chosen strain from each species. Effectors are labeled according to their molecular target (see below); effectors expected to induce rapid cell lysis (Am-1-3, Mur, Glc, PLA1-2, PLD; “fast-lysing”) are shaded in red; those expected to lyse cells more slowly (Tox43, Tse4, VasX, Tox46; “slow-lysing”) are shaded blue. A green marker signifies that a given strain has at least 1 fast-lysing effector in its repertoire. Each species in this dataset is represented by 1 strain only, and so these data are not representative of the full toxin repertoire of any given species. However, by comparing example strains across many species, one can robustly assess preference for known lytic toxins. Plotting data from all 474 species was not feasible in 1 figure, so we plot approximately half (222) of the strains here, retaining at least 1 representative from each genus. The full tree is available to download as a separate file (S1 Data). Original data are from LaCourse and colleagues [40]; reanalyzed data are available from dx.doi.org/10.6084/m9.figshare.11980491. Am-1-3, peptidoglycan amidases; Mur/Glc, peptidoglycan glucosidases; NCBI, National Center for Biotechnology Information; PLA1-2 and PLD, phospholipases; Tox43, DNases; Tox46; NAD(P)+ glycohydrolases; Tse4/VasX, pore-forming.