The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence

Abstract

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus's virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.

Fig 1. Representative T6SS gene clusters found in Vibrio coralliilyticus genomes.

The strain name, GenBank accession number, and the first and last locus tag are denoted on the left. Genes are denoted by arrows indicating the predicted direction of transcription. Encoded proteins or domains are denoted above the genes. T6SS, type VI secretion system.

Fig 2. Vibrio coralliilyticus T6SS1 and T6SS2 are regulated by environmental conditions.

Expression (cells) and secretion (media) of VgrG1 and Hcp2 from the 3 indicated V. coralliilyticus strains grown for 4 h at the indicated temperatures in “rich” LB containing 3% [wt/vol] NaCl (MLB) (A) or “poor” glycerol artificial seawater medium (B). (C) Comparison of VgrG1 and Hcp2 expression and secretion when V. coralliilyticus strains were grown at 28°C in “rich” or “poor” media. RNA polymerase sigma 70 (RNAp) was used as a loading and lysis control. Asterisks denote expected protein sizes. Results from a representative experiment out of at least 3 independent experiments are shown. LB, lysogeny broth; T6SS, type VI secretion system.

Fig 3. Vibrio coralliilyticus T6SS1 mediates interbacterial competition.

(A–C) Viability counts (CFU) of V. natriegens prey strains before (0 h) and after (4 h) co-incubation with the indicated V. coralliilyticus BAA-450 (A), OCN008 (B), or OCN014 (C) attacker strains on MLB plates at 28°C. The statistical significance between samples at the 4 h time point was calculated using an unpaired, two-tailed Student’s t test; ns, no significant difference (P > 0.05); WT, wild-type; DL, the assay’s detection limit. Data are shown as the mean ± SD; n = 3. The data shown are a representative experiment out of at least 3 independent experiments. The data underlying this figure can be found in S1 Data. CFU, colony-forming unit; T6SS, type VI secretion system.

Fig 4. Vibrio coralliilyticus T6SS2 mediates lethality in Artemia nauplii and in macrophages.

(A) Artemia nauplii were challenged with the indicated V. coralliilyticus OCN008 strains, and survival was assessed 40 to 56 h postinfection. Approximately 5 × 10⁷ bacteria were added to each well containing 2 nauplii. Data are shown as the mean ± SE of 4 biological replicates, each comprising 16 nauplii for every bacterial strain. The statistical significance between the WT and ΔtssM2 curves was calculated using the Log-rank (Mantel–Cox) test. (B–D) Assessment of cell death upon infection of BMDMs with the indicated V. coralliilyticus BAA-450 (B), OCN008 (C), or OCN014 (D) strains. Approximately 3.5 × 10⁴ BMDMs were seeded into 96-well plates in triplicates and infected with V. coralliilyticus strains at an MOI ~ 4. PI was added to the medium prior to infection, and its uptake kinetics were assessed using real-time microscopy. WT, wild-type. Results from a representative experiment out of at least 3 independent experiments are shown in B–D. The statistical significance between the WT and each of the mutants was calculated using a one-way ANOVA with Tukey’s multiple comparisons test using the area-under-the-curve values calculated for each sample; ns, no significant difference (P > 0.05). The data underlying this figure can be found in S2 Data. BMDM, bone marrow-derived macrophage; MOI, multiplicity of infection; PI, propidium iodide; T6SS, type VI secretion system.

Fig 5. Vibrio coralliilyticus T6SS1 effector repertoires.

Volcano plots summarizing the comparative proteomics of proteins identified in the media of the 3 indicated V. coralliilyticus strains with an active T6SS1 (WT, wild-type) or an inactive T6SS1 (Δhcp1), using LFQ. The average LFQ signal intensity difference between the WT and Δhcp1 strains is plotted against the -Log₁₀ of Student’s t test P-values (n = 3 biological replicates). Proteins that were significantly more abundant in the secretome of the WT strains (difference in average LFQ intensities > 1.6; P-value <0.02; with a minimum of 2 Razor unique peptides and Score >15) are denoted in green. The data underlying this figure can be found in S3 Data. LFQ, label-free quantification; T6SS, type VI secretion system.

Fig 6. Vibrio coralliilyticus T6SS2 effector repertoires.

Volcano plots summarizing the comparative proteomics of proteins identified in the media of the 3 indicated V. coralliilyticus strains with an active T6SS2 (WT, wild-type) or an inactive T6SS2 (ΔtssM2), using LFQ. The average LFQ signal intensity difference between the WT and ΔtssM2 strains is plotted against the -Log₁₀ of Student’s t test P-values (n = 3 biological replicates). Proteins that were significantly more abundant in the secretome of the WT strains (difference in average LFQ intensities >1.6; P-value <0.02; with a minimum of 2 Razor unique peptides and Score >15) are denoted in blue. The data underlying this figure can be found in S4 Data. LFQ, label-free quantification; T6SS, type VI secretion system.

Fig 7. Vibrio coralliilyticus T6SS2 effectors are toxic in eukaryotic cells.

(A) CoVes are toxic in yeast. Ten-fold serial dilutions of S. cerevisiae strains containing plasmids for the galactose-inducible expression of the indicated CoVes, or eGFP used as a negative control, were spotted on repressing (2% [wt/vol] glucose) or inducing (2% [wt/vol] galactose and 1% [wt/vol] raffinose) agar plates. eGFP, enhanced GFP. (B) CoVes are not toxic to bacteria. E. coli strains containing plasmids for the arabinose-inducible expression of the indicated, C-terminally FLAG-tagged CoVes, the V. campbellii antibacterial T6SS effector Rte1 used as a positive control, or an empty plasmid (Empty) were streaked onto repressing (0.4% [wt/vol] glucose) or inducing (0.001% [wt/vol] arabinose) agar plates. Results from a representative experiment out of at least 3 independent experiments are shown. T6SS, type VI secretion system.

Fig 8. The Vibrio coralliilyticus T6SS2 effector repertoire can be divided into core and accessory arsenals.

Distribution of T6SS2 CoVe1-9 effectors in RefSeq V. coralliilyticus genomes. The phylogenetic tree is based on a comparison of the codon sequences of 1,210 complete core genome proteins found in the indicated strains. The evolutionary history was inferred using the maximum likelihood method. Bootstrap values appear next to the corresponding branch as percent of 100 replicates. The data underlying this figure can be found in S5 Data. T6SS, type VI secretion system.