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A binary effector module secreted by a type VI secretion system

Abstract Gram‐negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Cargo effectors are secreted by binding noncovalently to the T6SS apparatus. Occasionally, effector secretion is assisted by an adaptor protein, although the adaptor itself is not secreted. Here, we report a new T6SS secretion mechanism, in which an effector and a co‐effector are secreted together. Specifically, we identify a novel periplasm‐targeting effector that is secreted together with its co‐effector, which contains a MIX (marker for type sIX effector) domain previously reported only in polymorphic toxins. The effector and co‐effector directly interact, and they are dependent on each other for secretion. We term this new secretion mechanism “a binary effector module,” and we show that it is widely distributed in marine bacteria.

A B Figure EV2. Generating a vp1388 mutant with no polar effect on VP1390 expression.
A Expression of VP1390 and VP1388 from V. parahaemolyticus mutant strains. Samples were treated with 20 µM phenamil to activate surface sensing in media containing 3% NaCl at 30°C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VP1390 and VP1388. B The growth of V. parahaemolyticus mutants in media containing 3% NaCl at 30°C, as measured by OD 600 readings. Data are shown as the mean AE SD, n = 4 technical replicates. WT, wild-type.  The growth of V. parahaemolyticus mutants in media containing 3% NaCl at 30°C, as measured by OD 600 readings. Data are shown as the mean AE SD, n = 4 technical replicates. WT, wild-type. The experiment was performed three times with similar results; results of a representative experiment are shown.
A D B C E Figure EV4. VP1390 mediates toxicity in the periplasm.
A Viability counts of V. parahaemolyticus Δvp1389 prey before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl and 0.001% arabinose at 30°C. Prey cells contain an empty plasmid (pEmpty) or a plasmid for expression of C-terminal Myc-tagged VP1389 (pVP1389) or its truncated version lacking the first 17 amino acids (pVP1389 ΔSS ). Data are shown as the mean AE SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. Δhcp1 was used as a T6SS1 À control strain. B The arabinose-inducible expression of C-terminal Myc-tagged VP1389 or VP1389 ΔSS in V. parahaemolyticus Δvp1389 strains described in A was detected using a-Myc antibodies. Loading control (LC) is shown for total protein lysates. C The expression of C-terminal Myc-tagged sfGFP, VP1388, and VP1390 fused to an N-terminal PelB signal peptide ( peri sfGFP, peri VP1388, and peri VP1390, respectively) was detected upon expression from an arabinose-inducible plasmid in E. coli, using a-Myc antibodies. Loading control (LC) is shown for total protein lysates. D Toxicity in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control) or VP1390 fused to an N-terminal PelB signal peptide ( peri sfGFP or peri VP1390, respectively), or VP1390 ( cyto VP1390) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated. E The expression of C-terminal Myc-tagged proteins described in D was detected upon expression from an arabinose-inducible plasmid in E. coli, using a-Myc antibodies. Loading control (LC) is shown for total protein lysates.
Data information: The experiments were performed at least three times with similar results; results of a representative experiment are shown in each panel. In C and E, bands corresponding to the protein expressed at the expected size are denoted by red asterisks. Source data are available online for this figure. Figure EV5. Lysis of sensitive prey cells is preceded by a bleb originating from the septum.
Representative image from time-lapse microscopy of competition between V. parahaemolyticus Δhns (T6SS + /VP1390 + ) attackers and V. parahaemolyticus Δvp1389 prey cells that express GFP, as shown in Fig 5. Attacker and prey were mixed (2:1 ratio) and spotted on LB agarose pads supplemented with propidium iodide (PI; pink). Merging of the phase contrast, GFP (green) and PI (pink) channels, as well as the PI or GFP channels alone are shown. Scale bar = 2 µm.