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. 2017 Nov;18(11):1978-1990.
doi: 10.15252/embr.201744226. Epub 2017 Sep 14.

Type VI Secretion System MIX-effectors Carry Both Antibacterial and Anti-Eukaryotic Activities

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Free PMC article

Type VI Secretion System MIX-effectors Carry Both Antibacterial and Anti-Eukaryotic Activities

Ann Ray et al. EMBO Rep. .
Free PMC article

Abstract

Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti-eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine-like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX-effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti-eukaryotic effectors. One of these is a MIX-effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX-effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors.

Keywords: Vibrio; CNF1; bacterial competition; virulence.

Figures

Figure 1
Figure 1. T6SS clusters and modules identified in silico in Vpr

Genes are represented by arrows indicating direction of translation. Locus tags (vpr01s_xx_xxxxx) shown above. Encoded proteins and known domains shown below.

Figure 2
Figure 2. Vpr is bactericidal under warm marine‐like conditions

Viability counts of Escherichia coli prey before (0 h) and after (4 h) co‐culture with wild‐type Vpr or with no attacker (none) at indicated temperatures on media containing 1% (A) or 3% (B) NaCl.

Figure 3
Figure 3. T6SS1 mediates Vpr bactericidal activity

Viability counts of Escherichia coli (A, B) and Vibrio parahaemolyticus POR1/Δhcp1 (C) prey before (0 h) and after (4 h) co‐culture with indicated Vpr strains on media containing 3% NaCl at 30°C. Asterisks mark statistical significance between samples at 4‐h timepoint by unpaired, two‐tailed Student's t‐test (*< 0.05); n.s., no significant difference; WT, wild‐type.

Expression (Cells) and secretion (Media) of VgrG1 were detected by immunoblot using specific antibodies against VgrG1. Loading control (LC) is shown for total protein lysate.

Data information: In (B–D), attackers contain either an empty expression vector (Empty) or vector for arabinose‐inducible expression of TssG1 (pTssG1). Source data are available online for this figure.
Figure 4
Figure 4. The Vpr T6SS1 secretome

Schematic representation of putative Vpr T6SS1‐secreted proteins identified by comparative proteomics (see Table 1). Horizontal black bars are relative to protein length. MIX domains and domains identified in NCBI CDD are illustrated in color; domain names are inside or below the rectangles. Transmembrane helices (TM) identified in UniProt are illustrated as white ovals with dashed perimeters. DUF4150 belong to PAAR‐like superfamily; DUF2235 is a phospholipase.

Figure EV1
Figure EV1. Vpr01570 is a functional anti‐eukaryotic toxin

Confocal micrograph of HeLa cells transfected with vectors expressing the indicated proteins. Cells were stained for F‐actin and DNA using rhodamine‐phalloidin (red) and Hoechst stain (blue), respectively. Scale bar = 30 μm; sfGFP, superfolder green fluorescent protein. Arrowheads mark actin ruffles.

Growth of BY4741 yeast containing vectors for galactose‐inducible expression of indicated proteins on repressing (glucose) and inducing (galactose) plates. Yeast were spotted in 10‐fold serial dilutions. VopRΔ90/CA is a truncated and catalytically inactive form of the Vibrio parahaemolyticus type III effector VopR and serves as a non‐toxic control.

Immunoblot using anti‐GFP (C) or anti‐Myc (D) antibodies to verify expression of proteins used in (A) and (B), respectively. In (C), black arrow marks expected size of Vpr01570‐sfGFP fusions and white arrow marks expected size of sfGFP. In (D), black arrow marks the expected size of Vpr01570‐myc, and white arrow marks the expected size of VopRΔ90/CA‐eGFP fusion. LC, loading control.

Source data are available online for this figure.
Figure 5
Figure 5. The T6SS1 MIX‐effector Vpr01570 induces actin rearrangements in macrophages

Confocal micrograph of RAW 264.7 cells infected with indicated Vpr or avirulent Vibrio parahaeolyticus (V. para) strains for 4 h. Cells were stained for F‐actin and DNA using rhodamine‐phalloidin (yellow) and Hoechst stain (cyan), respectively. Top panels show bottom focal plane of the cells; lower panels show stacked z‐axis slices of the same field of view. White arrows mark free nuclei of lysed cells. Red arrows mark actin ruffles. Scale bar = 20 μm; WT, wild‐type.

Confocal micrograph of RAW 264.7 cells infected with indicated Vpr strains containing either an empty vector (Empty) or arabinose‐inducible vectors for expression of wild‐type Vpr1570 (pVpr1570) or C450S mutant form (pVpr1570CS) for 4 h at MOI = 25. Cells were stained for F‐actin using rhodamine‐phalloidin. F‐actin is color‐coded to show cell depth in the z‐axis. Scale bar = 20 μm; WT, wild‐type.

Quantification of percentage of infected cells presenting a flatten and ruffled morphology after 6 h infection with the indicated strains or addition of LPS (0.1 μg/ml). Results shown as mean percentage of cells in a field ± standard deviation (n = 3, minimum number of cells per field is 40; maximum number of cells per field is 87). Asterisks mark treatments that are significantly different from LPS treatment as determined by unpaired, two‐tailed Student's t‐test (*< 0.05). Experiment was performed at least twice and a representative result is shown.

Figure EV2
Figure EV2. Vpr‐induced morphological changes in macrophages begin 4 h post‐infection

Confocal micrograph of RAW 264.7 cells infected with Vpr Δvprh strain at MOI = 25 or treated with LPS (0.1 μg/ml) for the indicated times. Cells were stained for F‐actin using rhodamine‐phalloidin. Top panels show fluorescence images. F‐actin is color‐coded to show cell depth in the z‐axis. Bottom panels show DIC of same fields of view. Scale bar = 50 μm.

Quantification of percentage of infected cells presenting a flatten and ruffled morphology during Vpr infection or LPS treatment as in (A). Results shown as mean percentage of cells in a field ± standard deviation (n = 3, minimum number of cells per field is 41; maximum number of cells per field is 95). Experiment was performed three times, and a representative result is shown.

Figure EV3
Figure EV3. Vpr01570 is sufficient to induce actin ruffles in macrophages

Confocal micrograph of RAW 264.7 cells transfected with vectors expressing the indicated proteins. Cells were stained for F‐actin and DNA using rhodamine‐phalloidin (red) and Hoechst stain (blue), respectively. Top panels show a focal plane at the top of the cells; bottom panels show a focal plane located at the bottom of the cells. Scale bar = 20 μm; sfGFP, superfolder green fluorescent protein.

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