
==== Front
PLoS Biol
PLoS Biol
plos
PLOS Biology
1544-9173
1545-7885
Public Library of Science San Francisco, CA USA

10.1371/journal.pbio.3002734
PBIOLOGY-D-24-00845
Research Article
Biology and Life Sciences
Microbiology
Bacteriology
Bacterial Physiology
Secretion Systems
Biology and Life Sciences
Microbiology
Microbial Physiology
Bacterial Physiology
Secretion Systems
Medicine and Health Sciences
Pathology and Laboratory Medicine
Pathogens
Virulence Factors
Secretion Systems
Biology and Life Sciences
Organisms
Bacteria
Vibrio
Biology and Life Sciences
Genetics
Genomics
Medicine and Health Sciences
Pathology and Laboratory Medicine
Pathogens
Virulence Factors
Biology and Life Sciences
Marine Biology
Corals
Earth Sciences
Marine and Aquatic Sciences
Marine Biology
Corals
Biology and Life Sciences
Organisms
Eukaryota
Fungi
Yeast
Biology and Life Sciences
Physiology
Physiological Processes
Secretion
Biology and Life Sciences
Toxicology
Toxicity
Medicine and Health Sciences
Pathology and Laboratory Medicine
Toxicology
Toxicity
The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence
Vibrio coralliilyticus T6SS effector repertoires
Mass Shir Formal analysis Investigation Methodology Writing – original draft 1
Cohen Hadar Formal analysis Investigation Methodology Writing – review & editing 1
Podicheti Ram Formal analysis Resources Writing – review & editing 2
Rusch Douglas B. Formal analysis Resources Writing – review & editing 2
Gerlic Motti Funding acquisition Resources Writing – review & editing 1
Ushijima Blake Conceptualization Funding acquisition Resources Writing – review & editing 3
van Kessel Julia C. Conceptualization Funding acquisition Resources Writing – review & editing 4
Bosis Eran Formal analysis Funding acquisition Investigation Methodology Writing – review & editing 5
https://orcid.org/0000-0002-2009-9453
Salomon Dor Conceptualization Formal analysis Funding acquisition Supervision Writing – original draft 1 *
1 Department of Clinical Microbiology and Immunology, School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, Tel Aviv, Israel
2 Center for Genomics and Bioinformatics Indiana University, Bloomington, Indiana, United States of America
3 Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, North Carolina, United States of America
4 Department of Biology, Indiana University, Bloomington, Indiana, United States of America
5 Department of Biotechnology Engineering, Braude College of Engineering, Karmiel, Israel
Waldor Matthew K. Academic Editor
Brigham and Women’s Hospital, UNITED STATES OF AMERICA
The authors have declared that no competing interests exist.

* E-mail: dorsalomon@mail.tau.ac.il
3 9 2024
9 2024
3 9 2024
22 9 e300273420 3 2024
3 7 2024
© 2024 Mass et al
2024
Mass et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

Vibrio coralliilyticus is a pathogen of coral and shellfish that is becoming more virulent as ocean temperatures rise, causing significant damage. This study shows that V. coralliilyticus has two type VI secretion systems (T6SSs) that are activated at high temperatures; one targets bacterial competitors, while the other launches up to 9 novel toxins against eukaryotic hosts like shrimps.

http://dx.doi.org/10.13039/100000001 National Science Foundation IOS-2207168 van Kessel Julia C. http://dx.doi.org/10.13039/100000001 National Science Foundation IOS-2207169 Ushijima Blake http://dx.doi.org/10.13039/100006221 United States - Israel Binational Science Foundation 2021733 https://orcid.org/0000-0002-2009-9453
Salomon Dor http://dx.doi.org/10.13039/501100003977 Israel Science Foundation 1362/21 https://orcid.org/0000-0002-2009-9453
Salomon Dor http://dx.doi.org/10.13039/501100003977 Israel Science Foundation 1362/21 Bosis Eran http://dx.doi.org/10.13039/501100003977 Israel Science Foundation 2174/22 Gerlic Motti This project received funding from the National Science Foundation (https://www.nsf.gov) and US-Israel Binational Science Foundation (https://www.bsf.org.il) (NSF-BSF) under award numbers IOS-2207168 (JCVK), IOS-2207169 (BU), and 2021733 (DS), and from the Israel Science Foundation (www.isf.org.il; ISF grant number 1362/21 to DS and EB, and grant number 2174/22 to MG). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityRelevant data are within the paper and its Supporting Information files. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD049479.
Data Availability

Relevant data are within the paper and its Supporting Information files. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD049479.
==== Body
pmcIntroduction

The oceans are home to gram-negative marine bacteria of the genus Vibrio. These include many established and emerging pathogens that infect humans and marine animals [1,2]. In the past, human pathogenic vibrios were primarily associated with the warmer equatorial waters. Yet, in recent decades, they have spread to other regions, including the northern United States of America, Canada, and North Europe [3,4]. This spread correlates with rising ocean surface-level temperatures and disease outbreaks [5,6].

Corals are marine animals affected by rising ocean temperatures caused by climate change and the spread of vibrios [7–9]. They are ecologically and economically important because they provide diverse ecosystems used as habitats for various fish and invertebrates, as well as help to protect shorelines from storm surges and erosion [10]. The coral animal lives in a symbiotic relationship with photosynthetic endosymbiotic dinoflagellates and microbes (collectively called the coral holobiont) [11–14]. Vibrio coralliilyticus is a bacterial pathogen shown to be a cause of diseases resulting in bleaching or tissue loss in corals [9,15,16]. Among other coral pathogens [9], V. coralliilyticus stands out due to its wide geographic spread and broad range of reported hosts. Aside from corals, V. coralliilyticus is also responsible for mortalities in shellfish hatcheries [17].

The coral holobiont is affected by various environmental conditions, such as shifts in water temperature, pH, and nutrients. Elevated temperature is a key factor in many V. coralliilyticus infections because it increases the abundance and virulence of many V. coralliilyticus strains [14]. At temperatures below 23°C, V. coralliilyticus strains are predominantly not pathogenic [8]. However, the virulence of many strains increases when temperatures rise above 23°C [14,15,18]. In some cases, the symbiotic dinoflagellates are killed and coral bleaching occurs. With most pathogenic strains, shifts to >27°C result in coral tissue lysis and increased coral mortality [15]. Elevated temperatures are associated with the production of proteases and hemolysins, motility, antimicrobial resistance, and secretion systems in V. coralliilyticus [19]. In addition, the expression of toxR, a transcription regulator associated with virulence in other vibrios [20], correlates with increased temperature and was shown to contribute to V. coralliilyticus virulence [21]. These data provide strong evidence that temperature regulates virulence-associated genes in V. coralliilyticus. Nevertheless, it remains unclear how these factors contribute to pathogenicity and whether the same factors play a role in virulence towards different hosts.

Many vibrios employ a specialized toxin delivery mechanism, the type VI secretion system (T6SS), to manipulate their environment [22–29]. The T6SS is a proteinaceous apparatus that is assembled inside the bacterial cell: a sheath structure engulfs an inner tube made of stacked hexameric rings of Hcp proteins, which is capped by a spike comprising a VgrG trimer sharpened by a PAAR repeat-containing protein (hereafter referred to as PAAR) [30]. This tube-spike complex is decorated with toxic proteins, called effectors, that mediate the toxic activities of the T6SS [31–33]. Contraction of the sheath propels the tube-spike complex out of the cell, providing it with sufficient force to penetrate the membrane of a neighboring cell where effectors are deployed [34]. Whereas most T6SSs investigated to date mediate interbacterial competitions by delivering antibacterial effectors, a few T6SSs have been shown to target eukaryotes and mediate virulence [33,35,36]. In accordance, although most Vibrio T6SSs play a role in interbacterial competitions [24–28,37–39], we and others recently revealed Vibrio T6SSs and effectors that target eukaryotes, and we postulated that they play a role in virulence [22,40–45].

Several studies reported the temperature-dependent expression of T6SS components in V. coralliilyticus [19,42], suggesting that T6SSs play a role in the temperature-regulated transition to a pathogenic lifestyle. The antibacterial activity of one T6SS was previously demonstrated in 2 V. coralliilyticus strains [42,46]. However, the presence of other T6SSs in the V. coralliilyticus pan-genome, their role, regulation, effector repertoire, and contribution to virulence remain unknown. Here, we systematically analyzed the T6SSs in the V. coralliilyticus pan-genome and revealed 2 omnipresent systems. Using 3 V. coralliilyticus strains as model systems, we experimentally defined the environmental conditions regulating the activation of these 2 T6SSs. We also identified their function and effector repertoires. Importantly, we revealed 9 novel anti-eukaryotic effectors delivered by the V. coralliilyticus T6SS2, contributing to V. coralliilyticus virulence.

Results

Two T6SSs are omnipresent in Vibrio coralliilyticus strains

To identify the T6SSs found in the pan-genome of V. coralliilyticus, we retrieved the sequences of the core T6SS sheath component, TssB, from 31 available RefSeq V. coralliilyticus genomes (S1 Dataset) and analyzed their genomic neighborhoods. Our analyses revealed that all genomes harbor 2 conserved T6SSs, named T6SS1 and T6SS2 (Fig 1 and S2 Dataset), suggesting that these T6SSs play an important role in the V. coralliilyticus lifestyle. T6SS1 is similar to the previously investigated T6SS1 from V. parahaemolyticus [24,47], V. alginolyticus [26], and V. proteolyticus [28], sharing the same gene content and organization. We recently showed that this system mediates interbacterial competition in the V. coralliilyticus type strain BAA-450 and in strain OCN008 [42,46]. The synteny of the T6SS1 (Fig A in S1 Text) and T6SS2 (Fig B in S1 Text) gene clusters is similar in all strains, with the exception of the 5′ region of T6SS1 containing different versions of a predicted effector, co-effector, and immunity protein combinations [48], as previously observed in similar T6SS gene clusters in V. parahaemolyticus [49,50]. Two additional T6SSs, which we named T6SS3 and T6SS4, are each found in a single V. coralliilyticus genome (S2 Dataset). Notably, 2 genes encoding structural core components in T6SS4 appear to include frameshifts, and the gene cluster lacks a gene encoding the conserved T6SS core component, TssH (Fig 1). Therefore, it is possible that T6SS4 is not functional.

10.1371/journal.pbio.3002734.g001 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.

Environmental conditions regulate Vibrio coralliilyticus T6SSs

Because T6SS1 and T6SS2 are omnipresent in V. coralliilyticus, we set out to investigate their activation and function. First, we sought to determine whether T6SS1 and T6SS2 are regulated by environmental conditions regulating V. coralliilyticus virulence. To this end, we selected 3 representative V. coralliilyticus strains harboring both T6SSs: BAA-450 (the type strain), OCN008, and OCN014. These strains were isolated from different coral hosts and display different disease etiologies [16,51,52]. Strains BAA-450 and OCN014 have a temperature-dependent infection mode, and they become more virulent as temperatures rise above 23°C; the virulence of strain OCN008 does not significantly change from 23 to 27°C [16,18,21].

To determine whether the activation of T6SS1 and T6SS2 depends on temperature or nutrient availability, we monitored the expression and secretion of the conserved secreted T6SS structural components, VgrG1 and Hcp2 [23], respectively. Bacteria were grown in either rich (LB containing 3% [wt/vol] NaCl; MLB) or poor (glycerol artificial seawater; GASW) media and under a range of physiologically relevant temperatures that affect V. coralliilyticus pathogenicity: 19, 23, 28, and 31°C [16,21,51]. As shown in Fig 2A and 2B, we found that the activity of both T6SS1 and T6SS2 is temperature and media dependent. In rich media, both systems are active between 23 and 31°C; T6SS1 secretion peaks at 28°C, whereas T6SS2 secretion peaks at 31°C (Fig 2A). Notably, secretion via T6SS1 in strain OCN008 appears lower than in BAA-450 and OCN014. In poor media, T6SS1 secretion peaks at 23°C in all strains and is retained at higher temperatures only in strain BAA-450 (Fig 2B); T6SS2 secretion is only observed in strain BAA-450 at 23°C. Comparison between the activity of both systems in rich and poor media at 28°C revealed higher levels of expression and secretion in rich media (Fig 2C). Therefore, unless otherwise indicated, we performed subsequent analyses of T6SS1 and T6SS2 when V. coralliilyticus strains are grown in rich media at 28°C, conditions in which both systems are active in all 3 strains.

10.1371/journal.pbio.3002734.g002 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.

T6SS1 mediates interbacterial competitions

We previously reported that T6SS1 in strains BAA-450 and OCN008 mediates antibacterial activity during interbacterial competitions [42,46]. To determine whether this is also true for T6SS1 in strain OCN014 and whether T6SS2 also plays a role in interbacterial competition, we set out to monitor the outcome of interbacterial competitions using V. coralliilyticus strains in which the 2 T6SSs were inactivated, either individually or together. To this end, we first constructed V. coralliilyticus mutant strains in which we inactivated T6SS1 by deleting the gene encoding the conserved structural component Hcp1 (Δhcp1) and T6SS2 by deleting the gene encoding the conserved structural component TssM2 (ΔtssM2) (Fig C in S1 Text, panel A). These mutations did not affect bacterial growth (Fig C in S1 Text, panel B). When competed against a sensitive V. natriegens prey strain on rich media plates at 28°C, all 3 V. coralliilyticus strains killed the prey, evident by the decrease in prey viability during the 4 h of co-incubation with the wild-type V. coralliilyticus attackers (Fig 3). This killing was dependent on T6SS1, since its inactivation in the attacker strains by deleting hcp1 abolished the toxicity. The T6SS1-mediated killing was also apparent when V. coralliilyticus OCN008 was competed against V. alginolyticus or V. campbellii, 2 species that were previously isolated from corals [53] (Fig D in S1 Text). Inactivation of T6SS2 by deleting tssM2, either alone or in combination with an inactive T6SS1, had no effect on the observed antibacterial activity of V. coralliilyticus (Fig 3). Taken together, our results confirm that the V. coralliilyticus T6SS1 mediates antibacterial activity and suggest that T6SS2 does not play a role in interbacterial competition.

10.1371/journal.pbio.3002734.g003 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.

T6SS2 targets eukaryotes

Based on the above results, we hypothesized that T6SS2 mediates anti-eukaryotic activities. To investigate whether T6SS2 plays a role in bacterial virulence, we employed the saline lake-dwelling brine shrimp, Artemia salina, as an aquatic animal model [41,54,55]. Wild-type V. coralliilyticus OCN008 was lethal to Artemia nauplii (larvae), with a median survival of 53 h. Inactivation of T6SS2, either alone (ΔtssM2) or together with T6SS1 (Δhcp1/ΔtssM2), resulted in a significantly reduced lethality (median survival undefined or 56 h, respectively), whereas inactivation of T6SS1 (Δhcp1) had no effect (Fig 4A). These results reveal a role for the V. coralliilyticus T6SS2 in pathogenicity during infection of a eukaryotic host.

To further investigate the anti-eukaryotic activity of V. coralliilyticus T6SS2 in all 3 strains, we used real-time microscopy to monitor V. coralliilyticus-mediated cell death kinetics. To this end, we employed bone marrow-derived macrophages (BMDMs), which have been previously used as a model to monitor the toxic effects of another Vibrio T6SS [40]. Various levels of cell death were observed starting ~30 min after adding either of the wild-type V. coralliilyticus strains BAA-450, OCN008, or OCN014 (Fig 4B–4D). Remarkably, inactivation of T6SS2, either alone (ΔtssM2) or together with T6SS1 (Δhcp1/ΔtssM2), completely abrogated the V. coralliilyticus-mediated cell death, whereas inactivation of T6SS1 (Δhcp1) had either no effect (OCN008 and OCN014) or only a mild effect (BAA-450). These results support our hypothesis that V. coralliilyticus T6SS2 targets eukaryotes.

10.1371/journal.pbio.3002734.g004 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.

T6SS1 and T6SS2 secrete diverse effector arsenals

Next, we performed comparative proteomics analyses to reveal the V. coralliilyticus T6SS secretomes and identify the effectors that mediate the antibacterial and anti-eukaryotic activities described above. Using mass spectrometry, we compared the proteins secreted by the wild-type V. coralliilyticus strains BAA-450, OCN008, and OCN014 with those secreted by their isogenic mutants in which either T6SS1 or T6SS2 have been inactivated (Δhcp1 or ΔtssM2, respectively).

T6SS1 secretomes

We identified 11, 6, and 11 proteins that were significantly enriched in the secretomes of wild-type strains BAA-450, OCN008, and OCN014, respectively, compared to their T6SS1− (Δhcp1) mutants (Fig 5 and Table 1 and S3–S5 Dataset). These include the secreted tube-spike structural components Hcp1 (which was deleted to inactivate T6SS1), VgrG1, and PAAR-like proteins. Most of the additional proteins are predicted antibacterial or anti-eukaryotic effectors, or proteins encoded next to them, including: (i) homologs of previously described T6SS effectors, with predicted toxic domains that target the peptidoglycan (e.g., WP_006961156.1 and WP_006961879.1); (ii) proteins containing MIX domains, which are markers for T6SS effectors [47], with predicted nuclease or pore-forming toxic domains (e.g., WP_039951132.1 and WP_201765497.1); and (iii) proteins that have yet to be described as related to T6SSs, which were identified only in the T6SS1 secretome of strain OCN008 (e.g., WP_021456284.1 and WP_021455387.1, which is a DEAD/DEAH box helicase). In accordance with our observation that the T6SS1 appears less active in strain OCN008 compared to the 2 other V. coralliilyticus strains under the assay conditions (Fig 2A), the comparative proteomics intensity difference for the putative OCN008 T6SS1 effectors was low (Fig 5), suggesting that the latter type of proteins detected only in the OCN008 T6SS1 secretome may be false positives. As previously reported for similar T6SSs in other vibrios [26,28,47,56], some of the identified proteins are encoded within the T6SS1 gene cluster, whereas others are encoded in auxiliary or orphan operons. Moreover, predicted antibacterial effectors are encoded next to putative immunity genes. Taken together, these results support our findings that V. coralliilyticus T6SS1 plays a role in interbacterial competitions using antibacterial effectors. Interestingly, in each V. coralliilyticus strain, we also identified a secreted MIX domain-containing effector that we previously showed or hypothesized targets eukaryotes rather than bacteria (e.g., WP_006962196.1) [22]. This finding suggests that T6SS1 also plays a role in interactions with eukaryotes, even though our experiments did not reveal significant T6SS1-mediated anti-eukaryotic effects.

10.1371/journal.pbio.3002734.g005 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.

10.1371/journal.pbio.3002734.t001 Table 1 Vibrio coralliilyticus T6SS1 secretomes identified by comparative proteomics.

Predicted role	Predicted activity or domain	BAA-450	OCN008	OCN014	
Protein accession	Gene locus	Protein accession	Gene locus	Protein accession	Gene locus	
T6SS structural	Hcp	WP_006961152.1	VIC_RS16330	WP_006961152.1	G3U99_RS23805	WP_006961152.1	JV59_RS20030	
VgrG	WP_006961151.1	VIC_RS16325	N/D	N/D	WP_040121702.1	JV59_RS20025	
PAAR-like (DUF4150)	WP_039952215.1	VIC_RS19185	N/D	N/D	WP_040122118.1	JV59_RS22990	
PAAR-like (DUF4150)	UPI0001B95600 (annotated as a pseudogene in RefSeq)	VIC_RS08805	WP_021455353.1	G3U99_RS15395	WP_021455353.1	JV59_RS10110	
Antibacterial effector	VP1390-like	WP_006961156.1	VIC_RS16350	WP_021456525.1	G3U99_RS23785	WP_040121705.1	JV59_RS20045	
Lysozyme-like	WP_006961879.1	VIC_RS19190	N/A	N/A	WP_040122119.1	JV59_RS22995	
MIX domain; TMs	WP_201765497.1	VIC_RS12080	N/D	N/D	WP_235284713.1	JV59_RS24085	
MIX domain; Pyocin_S; Colicin E9-like nuclease	WP_039951132.1	VIC_RS01010	N/A	N/A	N/A	N/A	
MIX domain; Colicin A-like pore-forming	N/A	N/A	N/A	N/A	WP_052131801.1	JV59_RS24930	
Unknown	N/A	N/A	WP_021456284.1	G3U99_RS12660	N/A	N/A	
Anti-eukaryotic effector	MIX domain	WP_006962196.1	VIC_RS20535	N/A	N/A	N/A	N/A	
MIX domain	N/A	N/A	WP_021456290.1	G3U99_RS26335	WP_235284669.1	JV59_RS27320	
Effector accessory	MIX domain-containing co-effector	WP_006961158.1	VIC_RS16360	N/D	N/D	WP_040121707.1	JV59_RS20055	
Encoded upstream of anti-eukaryotic MIX domain-containing effector	WP_006962197.1	VIC_RS20540	N/D	N/D	WP_038511497.1	JV59_RS07245	
Unknown	DEAD/DEAH box helicase	N/D	N/D	WP_021455387.1	G3U99_RS17670	N/A	N/A	
N/A, no homolog is encoded in the genome; N/D, a homolog is encoded in the genome but not detected in the mass spectrometry analysis; TM, transmembrane helix (according to phobius).

T6SS2 secretomes

We identified 10, 9, and 6 proteins that were significantly enriched in the secretomes of wild-type strains BAA-450, OCN008, and OCN014, respectively, compared to their T6SS2− (ΔtssM2) mutants (Fig 6 and Table 2 and S3–S5 Dataset). These include the secreted tube-spike structural components Hcp2 and VgrG2. We predict that all the other identified, nonstructural proteins, which are encoded outside the T6SS2 gene cluster (Fig E in S1 Text), are novel anti-eukaryotic effectors (excluding the phage shock protein, WP_021456780.1, which is probably a phage protein and not a T6SS effector). In support of this prediction, none of these proteins is encoded next to a gene that could encode for a cognate immunity protein. Moreover, some are similar to previously described virulence toxins, such as WP_006960006.1 containing a predicted YopT-like cysteine protease domain (YopT is a type III secretion system virulence effector from Yersinia [57]). No putative effectors have a predicted signal peptide for the Sec or Tat secretion systems that could account for their secretion, according to SignalP 6.0 [58] analyses.

10.1371/journal.pbio.3002734.g006 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.

10.1371/journal.pbio.3002734.t002 Table 2 Vibrio coralliilyticus T6SS2 secretomes identified by comparative proteomics.

Predicted role	Predicted activity or domain	BAA-450	OCN008	OCN014	
Protein accession	Gene locus	Protein accession	Gene locus	Protein accession	Gene locus	
T6SS structural	Hcp	WP_006962090.1	VIC_RS20130	WP_006962090.1	G3U99_RS13135	WP_006962090.1	JV59_RS07745	
VgrG	WP_006962095.1	VIC_RS20150	WP_021458175.1	G3U99_RS13115	WP_038511668.1	JV59_RS07720	
Anti-eukaryotic effector	CNF-likea (CoVe1)	WP_006957348.1	VIC_RS01360	WP_006957348.1	G3U99_RS07885	WP_006957348.1	JV59_RS02730	
(p)ppGpp synthetase / hydrolasea (CoVe2)	WP_006959328.1	VIC_RS09310	WP_021457154.1	G3U99_RS15865	WP_021457154.1	JV59_RS10570	
Cysteine peptidasea (CoVe3)	WP_006959836.1	VIC_RS11210	N/D	N/D	N/D	N/D	
peptidase_C58-like super familyb; TM (CoVe4)	WP_006960006.1	VIC_RS11685	WP_006960006.1	G3U99_RS19905	WP_006960006.1	JV59_RS23695	
Unknown (CoVe5)	WP_006961218.1	VIC_RS16620	WP_165579899.1	G3U99_RS23535	WP_040121739.1	JV59_RS20315	
ADP-ribosyltransferaseb (CoVe6)	WP_006961766.1	VIC_RS18765	WP_021457687.1	G3U99_RS21080	N/A	N/A	
Peptidase_26-likeb (CoVe7)	WP_006962231.1	VIC_RS20705	N/D	N/D	N/D	N/D	
TM (CoVe8)	WP_006962597.1	VIC_RS22130	N/D	N/D	N/D	N/D	
Unknown (CoVe9)	N/A	N/A	WP_141650583.1	G3U99_RS19765	N/A	N/A	
Unknown	Phage shock protein PspA	N/D	N/D	WP_021456780.1	G3U99_RS10060	N/D	N/D	
N/A, no homolog is encoded in the genome; N/D, a homolog is encoded in the genome but not detected in the mass spectrometry analysis; TM, transmembrane helix (according to phobius).

a According to HHpred.

b According to NCBI CDD.

T6SS2 effectors are novel anti-eukaryotic toxins

Since most of the T6SS1 effectors we identified are homologs of previously described effectors, we focused on the novel T6SS2 effectors for subsequent analyses. Altogether, the identified V. coralliilyticus T6SS2 effector repertoire comprises 9 putative novel effectors, which we named Coralliilyticus Virulence effector 1 to 9 (CoVe1-9): CoVe1, 2, 4, and 5 were identified in the secretomes of all 3 strains; CoVe6 was identified in the secretomes of BAA-450 and OCN008; CoVe3, 7, and 8 were identified only in the secretome of BAA-450, and CoVe9 was identified only in the secretome of OCN008 (Table 2).

Six of the 9 CoVes contain domains with predicted toxic activities (Table 2), including peptidase [57], ADP-ribosyltransferase [59], cytotoxic necrotizing factor (CNF)-like deamidase [60], and (p)ppGpp synthetase/hydrolase [61]. However, CoVe5, 8, and 9 sequence analyses did not reveal significant similarity to any previously investigated toxin, suggesting that they harbor novel toxic domains.

We sought to investigate these putative effectors. First, we set out to further validate their T6SS2-dependent secretion using a standard secretion assay. To this end, we cloned the 9 putative effectors (CoVe1-8 from strain BAA-450 and CoVe9 from strain OCN008) into an arabinose-inducible expression plasmid, fused to a C-terminal FLAG tag, and monitored their secretion to the media from V. coralliilyticus strains. As shown in Fig F in S1 Text, T6SS2-dependent secretion of all CoVes, except CoVe3, was evident upon ectopic overexpression from a plasmid in their respective encoding V. coralliilyticus strain. Since CoVe3 T6SS2-dependent secretion was observed in the more sensitive comparative proteomics approach when endogenously expressed from the bacterial chromosome (Fig 6), it is possible that its overexpression from a plasmid hampered the secretion; alternatively, the C-terminal tag that we added to allow CoVe immunoblot detection may have interfered with CoVe3 secretion.

Next, we tested our hypothesis that these novel effectors target eukaryotes. In support of this hypothesis, we found that all 9 effectors are toxic when ectopically expressed from a galactose-inducible plasmid in a eukaryotic heterologous model organism, the yeast Saccharomyces cerevisiae [62,63] (Fig 7A). In contrast, these effectors were not toxic when expressed from an arabinose-inducible plasmid in E. coli, used as a surrogate model bacterium (Figs 7B and G in S1 Text). These results indicate that T6SS2 secretes an arsenal of novel effectors with anti-eukaryotic activities.

10.1371/journal.pbio.3002734.g007 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.

T6SS2 effectors are differentially distributed in Vibrio coralliilyticus genomes

We and others previously showed that T6SS effector repertoires can be divided into core effectors present in all strains harboring the system and accessory effectors encoded only by a subset of strains [25,64,65]. Therefore, we sought to determine the distribution of CoVes in V. coralliilyticus genomes. Interestingly, 7 of the 9 CoVes are found in all available RefSeq V. coralliilyticus genomes (Fig 8 and S6 Dataset). We propose that these 7 CoVes constitute the core effector repertoire of the V. coralliilyticus T6SS2. In contrast, 2 effectors, CoVe6 and CoVe9, are found only in a subset of strains, suggesting that they are part of the accessory T6SS2 effector repertoire. Interestingly, homologs of CoVe2 and CoVe8 are also found in all V. coralliilyticus genomes (Fig E in S1 Text and S6 Dataset). Even though these homologs were not identified in our comparative proteomics analyses, it is possible that they are also T6SS2 effectors.

10.1371/journal.pbio.3002734.g008 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.

Discussion

V. coralliilyticus is a pathogen that inflicts devastating ecological and economic losses. Although environmental conditions, such as high temperatures, have been associated with increased virulence and a pathogenic lifestyle, the virulence factors it uses remain poorly understood. Here, we systematically analyzed the T6SSs in the V. coralliilyticus pan-genome. We revealed 2 omnipresent T6SSs, T6SS1 and T6SS2, which are regulated by temperature and appear to contribute to V. coralliilyticus virulence. Whereas T6SS1 mediates antibacterial toxicity and thus possibly contributes to host colonization indirectly, T6SS2 secretes an array of novel anti-eukaryotic effectors and appears to play a direct role in virulence.

T6SS1 plays a role in interbacterial competition, possibly contributing to the elimination of commensal microbiota during host colonization. The T6SS1 effectors identified in our comparative proteomics analyses are homologs of effectors previously reported in similar T6SSs of other vibrios [22,28,47,48] or that have been predicted based on the presence of the MIX domain that defines a widespread class of polymorphic T6SS effectors [22,47]. Therefore, we did not investigate these effectors further in this work. Notably, we observed a mild effect of T6SS1 inactivation on the toxicity of the BAA-450 strain toward BMDMs, suggesting that T6SS1 may exert some effect also on eukaryotic cells. This could be mediated by the anti-eukaryotic MIX effector identified in the comparative proteomics analysis. However, we did not observe a similar effect with strains OCN008 and OCN014, even though they, too, secrete a predicted anti-eukaryotic MIX effector (Table 1).

T6SS2 secretes an array of anti-eukaryotic effectors and mediates toxicity during infection of a model host, Artemia nauplii, and during infection of macrophages. Because T6SS2 is induced at high temperatures, in correlation with the onset of V. coralliilyticus virulence, we propose that it plays a role in the colonization and toxicity towards its natural hosts, coral and shellfish larvae. In future work, we will investigate the contribution of T6SS2 to V. coralliilyticus’s virulence in these natural hosts, and we will also determine whether it targets the coral itself or its endosymbiotic dinoflagellates.

Notably, only a few anti-eukaryotic T6SS effectors are known [35,36]. Although we recently revealed anti-eukaryotic effectors in vibrios, belonging to the RIX effector class [42], the CoVes reported here do not belong to any known polymorphic effector class and appear to be new T6SS effectors that have not been previously described. Even though their activities and cellular targets remain to be investigated, 6 CoVes harbor putative catalytic domains that have previously been implicated in virulence. Future investigations will reveal their mechanism of action and targets inside eukaryotic cells, as well as their contribution to virulence.

Although we did not observe T6SS2 secretion in the poor GASW media in V. coralliilyticus OCN008, this system contributed to this strain’s virulence during the infection of Artemia nauplii. Artemia nauplii were grown in poor media (i.e., Instant Ocean), yet the infection likely originated in the gastrointestinal tract of the animal, where nutrients may be more readily available. Host factors found in the animal gut may have also contributed to the activation of the T6SS during infection.

Notably, our results suggest that additional factors contribute to the virulence toward Artemia, since inactivation of T6SS2 did not completely abolish toxicity in this host. Interestingly, the results of the BMDM infection assays indicate that the V. coralliilyticus T6SS2 is also active at high temperatures of 37°C, which are infrequent in marine environments, and in low salinity media. It appears that under these conditions, the anti-eukaryotic toxicity of V. coralliilyticus strains is mediated predominantly by T6SS2, since its inactivation abrogated toxicity toward BMDMs. This observation suggests that V. coralliilyticus T6SS2 is functional under a wide temperature range and can overcome immune cells.

In conclusion, we find a new T6SS that specifically targets eukaryotes, and we identify its effector arsenal. Taken together with recent findings of vibrios that use T6SSs to target eukaryotes [22,40–45] and of widespread Vibrio-encoded anti-eukaryotic T6SS effectors [42], our results suggest that T6SSs should be considered as potential virulence factors. These findings shed light on the molecular mechanisms that govern the connection between rising seawater temperatures and V. coralliilyticus virulence.

Materials and methods

Strains and media

For a complete list of strains used in this study, see Table A in S1 Text. Escherichia coli strain DH5α (λ-pir) was grown in 2xYT broth (1.6% [wt/vol] tryptone, 1% [wt/vol] yeast extract, and 0.5% [wt/vol] NaCl) or on lysogeny broth (LB) agar plates (1.5% [wt/vol]) at 37°C. The media were supplemented with chloramphenicol (10 μg/ml) to maintain plasmids when needed. To repress expression from arabinose-inducible Pbad promoters, 0.4% (wt/vol) D-glucose was added to the media. To induce expression from Pbad, L-arabinose was added to the media at 0.001 or 0.1% (wt/vol), as indicated.

V. coralliilyticus strains ATCC BAA-450, OCN008, and OCN014, and their derivatives were grown in Marine Lysogeny broth (MLB; LB containing 3% [wt/vol] NaCl) or on GASW-Tris agar plates (20.8 [g/l] NaCl, 0.56 [g/l] KCl, 4.8 [g/l] MgSO4·7H2O, 4 [g/l] MgCl2·6H2O, 0.01 [g/l] K2HPO4, 0.001 [g/l] FeSO4·7H2O, 2 [g/l] Instant Ocean sea salts, 6.33 [g/l] Tris base [C4H11NO3], 4 [g/l] tryptone, 2 [g/l] yeast extract, 0.2% [vol/vol] glycerol, and 1.5% [wt/vol] agar; pH was adjusted to 8.3 with HCl) at 30°C. For colony selection after plasmid conjugation (see below), V. coralliilyticus was grown on TCBS agar (Millipore, #86348) plates. L-arabinose (0.01% [wt/vol]) was added to the media to induce expression from Pbad.

Vibrio natriegens ATCC 14048 were grown on Marine Minimal Media (MMM) agar plates (2% [wt/vol] NaCl, 0.4% [wt/vol] galactose, 5 mM MgSO4, 7 mM K2SO4, 77 mM K2HPO4, 35 mM KH2PO4, 2 mM NH4Cl, and 1.5% [wt/vol] agar) at 30°C. The media were supplemented with chloramphenicol (10 μg/ml) to select for or maintain plasmids when necessary.

Saccharomyces cerevisiae were grown in Yeast Extract–Peptone–Dextrose broth (YPD; 1% [wt/vol] yeast extract, 2% [wt/vol] peptone, and 2% [wt/vol] glucose) or on YPD agar plates (2% [wt/vol]) at 30°C. Yeast containing plasmids that provide prototrophy to leucine were grown in Synthetic Dropout media (SD; 6.7 [g/l] yeast nitrogen base without amino acids, 1.4 [g/l] yeast synthetic dropout medium supplement (Sigma)) supplemented with histidine (2 [ml/l] from a 1% [wt/vol] stock solution), tryptophan (2 [ml/l] from a 1% [wt/vol] stock solution), uracil (10 [ml/l] from a 0.2% [wt/vol] stock solution), and glucose (4% [wt/vol]). For galactose-inducible expression from a plasmid, cells were grown in SD media or on SD agar plates supplemented with galactose (2% [wt/vol]) and raffinose (1% [wt/vol]).

Plasmid construction

For a complete list of plasmids used in this study, see Table B in S1 Text. For a complete list of primers used in this study, see Table C in S1 Text. To enable strong, arabinose-inducible protein expression in V. coralliilyticus, we constructed the plasmid pKara1. To this end, we amplified the region between the araC cassette and rrnB T1 terminator, including a C-terminally FLAG-tagged sfGFP gene, from the plasmid psfGFP [48], and introduced it 220 bp upstream of the gene encoding the fluorescent protein DsRed in pVSV208 [66], using the Gibson assembly method.

For expression in bacteria, the coding sequences (CDS) of the indicated genes of interest were amplified by PCR from the respective genomic DNA of the encoding bacterium. Next, amplicons were inserted into the multiple cloning site (MCS) of pBAD33.1F, or in place of the sfGFP gene within pKara1, using the Gibson assembly method [67], in-frame with the C-terminal FLAG tag. Plasmids were introduced into E. coli DH5α (λ-pir) by electroporation and into vibrios via conjugation. Transconjugants were selected on TCBS agar (Millipore) plates supplemented with chloramphenicol.

For galactose-inducible expression in yeast, genes were inserted into the MCS of the shuttle vector pGML10 (Riken) using the Gibson assembly method, in-frame with a C-terminal Myc tag. Yeast transformations were performed using the lithium acetate method, as described previously [68].

Construction of deletion strains

To delete genes in V. coralliilyticus BAA-450, OCN008, and OCN014, 1 kb sequences upstream and downstream of each gene to be deleted were cloned together into the MCS of pDM4, a CmROriR6K suicide plasmid. The pDM4 constructs were transformed into E. coli DH5α (λ-pir) by electroporation and then conjugated into V. coralliilyticus strains. Transconjugants were selected on TCBS agar plates supplemented with chloramphenicol and then counter-selected on agar plates containing 15% (wt/vol) sucrose for loss of the sacB-containing plasmid. Deletions were confirmed by PCR.

Vibrio protein secretion assays

Secretion assays were performed as previously reported [24], with minor modifications. V. coralliilyticus strains were grown for 16 h in MLB supplemented with antibiotics to maintain plasmids when necessary. Bacterial cultures were diluted 4-fold in fresh media and incubated for 2 additional hours at 28°C. Then, the cultures were normalized to an optical density at 600 nm (OD600) of 0.18 in 5 ml of MLB or GASW media, as indicated. When protein expression from an arabinose-inducible plasmid was required, the media were supplemented with chloramphenicol and 0.01% (wt/vol) L-arabinose. The cultures were then incubated with continuous shaking (220 rpm) at 19°C, 23°C, 28°C, or 31°C, as indicated, for 4 h. For expression fractions, 0.5 OD600 units were harvested, and cell pellets were resuspended in 30 μl of 2× Tris-glycine SDS sample buffer (Novex, Life Sciences) with 5% (vol/vol) β-mercaptoethanol. For secretion fractions, supernatant volumes equivalent to 5 OD600 units were filtered (0.22 μm), and proteins were precipitated using the deoxycholate and trichloroacetic acid method [69]. The precipitated proteins were washed twice with cold acetone and air-dried before being resuspended in 20 μl of 100 mM Tris-Cl (pH = 8.0) and 20 μl of 2× Tris-glycine SDS sample buffer containing 5% (vol/vol) β-mercaptoethanol. Protein samples were incubated at 95°C for 10 min before being resolved on TGX Stain-free gels (Bio-Rad). The proteins were transferred onto 0.2 μm nitrocellulose membranes using Trans-Blot Turbo Transfer (Bio-Rad), following the manufacturer’s protocol. Membranes were then immunoblotted with custom-made α-Hcp2 (GenScript; polyclonal antibodies raised in rabbits against the peptides CGEGGKIEKGPEVGF or CVMTKPNREGSGADP; the latter was used only in the experiment shown in Fig 2A), Custom-made polyclonal α-VgrG1 [50], monoclonal α-FLAG (Sigma-Aldrich, F1804), or Direct-Blot HRP anti-E. coli RNA polymerase sigma 70 (mouse mAb #663205; BioLegend; referred to as α-RNAP) antibodies at a dilution of 1:1,000. Protein signals were detected using enhanced chemiluminescence (ECL) reagents with a Fusion FX6 imaging system (Vilber Lourmat).

Mass spectrometry analyses

Sample preparations for mass spectrometry were performed as described in the “Vibrio protein secretion assays” section. After the acetone wash step, samples were shipped to the Smoler Proteomics Center at the Technion, Israel, for analysis. Precipitated proteins were washed twice in 80% (vol/vol) cold acetone. The protein pellets were dissolved in 8.5 M Urea, 400 mM ammonium bicarbonate, and 10 mM DTT. Protein concentrations were estimated using the Bradford assay. The proteins were reduced at 60°C for 30 min and then modified with 35.2 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min at room temperature in the dark. The proteins were digested overnight at 37°C in 1.5 M urea and 66 mM ammonium bicarbonate with modified trypsin (Promega) at a 1:50 (M/M) enzyme-to-substrate ratio. An additional trypsinization step was performed for 4 h. The resulting tryptic peptides were analyzed by LC-MS/MS using Q Exactive HF mass spectrometer (Thermo) fitted with a capillary HPLC (Evosep). The peptides were loaded onto a 15 cm ID 150 1.9-micron (Batch no. E1121-3-24) column of Evosep. The peptides were eluted with the built-in Xcalibur 15 SPD (88 min) method. Mass spectrometry was performed in a positive mode using repetitively full MS scan (m/z 350 to 1,200) followed by High Energy Collision Dissociation (HCD) of the 20 most dominant ions selected from the full MS scan. A dynamic exclusion list was enabled with exclusion duration of 20 s.

The mass spectrometry data were analyzed with the MaxQuant software 2.1.1.0 (www.maxquant.org) using the Andromeda search engine [70] against the relevant V. coralliilyticus strains from the Uniprot database, with a mass tolerance of 4.5 ppm for the precursor masses and 4.5 ppm for the fragment ions. Peptide- and protein-level false discovery rates (FDRs) were filtered to 1% using the target-decoy strategy. The protein table was filtered to eliminate identities from the reverse database and common contaminants. The data were quantified by label-free analysis using the same software, based on extracted ion currents (XICs) of peptides, enabling quantitation from each LC/MS run for each peptide identified in any of the experiments. Statistical analyses of the identification and quantization results were done using the Perseus 1.6.7.0 software [71]. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via PRIDE [72].

Bacterial competition assays

Bacterial competition assays were performed as previously described [24], with minor modifications. Attacker and prey strains were grown for 16 h in appropriate media. In the morning, V. coralliilyticus attacker strains were diluted 1:10 into fresh media and incubated for an additional hour at 28°C. Attacker and prey cultures were then normalized to an OD600 of 0.5 and mixed at a 4∶1 (attacker:prey) ratio in triplicate. Next, the mixtures were spotted (25 μl) on MLB agar competition plates and incubated at 28°C for 4 h. The colony-forming units (CFU) of the prey strains at t = 0 h were determined by plating tenfold serial dilutions on selective media plates. After 4 h of co-incubation on competition plates, the bacteria were harvested, and the CFUs of the surviving prey strains were determined as described above. Prey strains contained a pBAD33.1 [73] (V. natriegens and V. alginolyticus) or pVSV208 [66] (V. campbellii) plasmid to allow selective growth on plates containing chloramphenicol.

Vibrio coralliilyticus growth assays

Triplicates of V. coralliilyticus cultures grown for 16 h were normalized to OD600 = 0.01 in MLB and transferred to a 96-well plate (200 μl per well). The 96-well plate was incubated in a microplate reader (BioTek SYNERGY H1) at 28°C with continuous shaking (205 cpm). Growth was measured as OD600 in 10-min intervals.

Artemia infection assays

Artemia infection assays were performed as previously reported [41], with minor modifications. Artemia salina eggs (Artemio Pur; JBL) were incubated in deionized distilled water containing chloramphenicol (10 μg/ml), kanamycin (100 μg/ml), and ampicillin (100 μg/ml) at 28°C with continuous rotation for an hour. The eggs were washed 4 times with Instant Ocean solution (3.3% [wt/vol]; Aquarium Systems) and then incubated for 24 h with continuous rotation at 28°C. Hatched Artemia nauplii were transferred into sterile 48-well plates (2 nauplii per well in 400 μl Instant Ocean). Approximately 5 × 107 bacteria were added to each well, and the plates were incubated at 28°C under 12-h light and dark cycles. Artemia survival was determined at the indicated time points postinfection. An Artemia nauplius that did not move for 10 s was defined as nonviable. Each bacterial strain was added to 8 wells (16 nauplii). Survival results are provided as grouped data from 4 independent experiments. Percent survival was calculated as surviving subjects out of the subjects at risk for each time point.

BMDM infection assays

Bone marrow cells from 6- to 8-week-old mice were isolated, and BMDMs were obtained after a 7-day differentiation, as previously described [74]. V. coralliilyticus strains were grown for 16 h in MLB. In the morning, bacterial cultures were diluted tenfold into fresh media and incubated for an additional hour at 28°C. Approximately 3.5 × 104 BMDMs were seeded into 96-well plates in triplicates in 1% (vol/vol) FBS and penicillin-streptomycin-free DMEM media and then infected with the indicated V. coralliilyticus strains at a multiplicity of infection (MOI) ~ 4. Plates were centrifuged for 5 min at 400 × g. Propidium iodide (PI; 1 μg/ml) was added to the medium 30 min prior to infection, and its uptake kinetics were assessed every 15 min using real-time microscopy (Incucyte SX5) during incubation at 37°C. The data were analyzed using the Incucyte SX5 analysis software and exported to Graphpad PRISM. Normalization was performed according to the maximal PI-positive object count to calculate the percentage of dead cells [74].

Yeast toxicity assays

Toxicity assays in yeast were performed as previously described [68]. Briefly, yeast cells were cultured for 16 h in SD media supplemented with 4% glucose (wt/vol). Yeast cultures were washed twice with sterile deionized distilled water and normalized to an OD600 of 1.0 in sterile deionized water. Then, 10-fold serial dilutions were spotted onto SD agar plates containing 4% (wt/vol) glucose (repressing plates) or 2% (wt/vol) galactose and 1% (wt/vol) raffinose (inducing plates). The plates were incubated at 28°C for 2 days.

Protein expression in E. coli

Overnight-grown bacterial cultures of E. coli DH5α (λ-pir) strains carrying pBAD33.1 arabinose-inducible expression plasmids were grown in 2xYT broth supplemented with chloramphenicol. Bacterial cultures were normalized to an OD600 = 0.5 in 3 ml fresh 2xYT with chloramphenicol and incubated with continuous shaking (220 rpm) at 37°C for 2 h. Then, L-arabinose was added to a final concentration of 0.1% (wt/vol) to induce protein expression, and the cultures were incubated for 2 additional hours. Cells equivalent to 0.5 OD600 units were harvested, and their pellets were resuspended in 50 μl of 2× Tris-glycine SDS sample buffer (Novex, Life Sciences) supplemented with 5% (vol/vol) β-mercaptoethanol. Subsequently, the samples were boiled at 95°C for 10 min and resolved on a TGX Stain-free gel (Bio-Rad) for SDS-PAGE analysis. The proteins were transferred onto nitrocellulose membranes, which were then immunoblotted with α-FLAG (Sigma-Aldrich, F1804) antibodies at a 1:1,000 dilution. Finally, protein signals were detected using ECL in a Fusion FX6 imaging system (Vilber Lourmat). The loading control for total protein lysates was visualized as the fluorescence of activated trihalo compounds found in the gel.

E. coli toxicity assays

To determine the toxicity of V. coralliilyticus proteins in bacteria, E. coli DH5α (λ-pir) strains carrying pBAD33.1 arabinose-inducible expression plasmids were streaked onto LB agar plates supplemented with chloramphenicol and either 0.4% (wt/vol) glucose (repressing plates) or 0.001% (wt/vol) L-arabinose (inducing plates). Plates were incubated for 16 h at 37°C.

Identifying T6SS gene clusters in Vibrio coralliilyticus

A local database containing the RefSeq bacterial nucleotide and protein sequences was generated (last updated on August 21, 2023). V. coralliilyticus genomes under NCBI Taxonomy ID = 190893 were retrieved from the local database, and OrthoANI [75] was performed as described previously [76]. The V. coralliilyticus strain SCSIO 43001 genome (assembly accession GCF_024449095.1) was removed from the data set because it showed OrthoANI values <95%. The V. coralliilyticus strain RE22 (assembly accession GCF_001297935.1) was removed because an updated version of strain RE22 was found (assembly accession GCF_003391375.1).

The presence of T6SS gene clusters in V. coralliilyticus genomes was determined by following a two-step procedure described previously [56]. Briefly, in the first step, BLASTN was employed to align V. coralliilyticus nucleotide sequences against the nucleotide sequences of representative T6SS clusters (Fig 1 and S2 Dataset). The best alignments for each nucleotide accession number were saved. In the second step, a 2D matrix was generated for each T6SS gene cluster. The matrices were filled in with the percent identity values based on the positions of the alignments from the first step. The overall coverage was calculated for each T6SS gene cluster in each genome. V. coralliilyticus genomes with at least 70% overall coverage of a T6SS gene cluster were regarded as containing that T6SS gene cluster (S2 Dataset). Comparative gene cluster analyses to determine the synteny of T6SS1 and T6SS2 were performed using CLINKER [77]. GenBank files used for CLINKER analyses were retrieved from NCBI.

Identifying effector homologs in Vibrio coralliilyticus genomes

BLASTP was employed to identify homologs of the T6SS2 effectors in V. coralliilyticus genomes, as described previously [25]. The amino acid sequences of new CoVes from strains BAA-450 (WP_006957348.1, WP_006959328.1, WP_006959836.1, WP_006960006.1, WP_006961218.1, WP_006961766.1, WP_006962231.1, and WP_006962597.1) and OCN008 (WP_141650583.1) were used as queries. The E-value threshold was set to 10−12, and the coverage was set to 70% based on the length of the query sequences.

Constructing a phylogenetic tree

The 1,445 core gene BUSCO definitions from vibrionales_odb10 were searched against the protein sequences for the 31 V. coralliilyticus strains using BUSCO version 4.0.5 [78]. Of these, 1,210 were found to be complete and non-duplicated in all 31 strains. Multiple sequence alignments were generated from the protein sequences homologous to each common core BUSCO using MUSCLE ver. 3.8.31 [79] with parameters: “-diags -sv -distance1 kbit20_3”, which were then converted to nucleotide space by substituting each amino acid with the corresponding codon sequence from the associated coding sequences. The codon alignments were concatenated together, and a best scoring maximum likelihood tree was drawn using RAxML version 8.2.12 [80] along with bootstrapping from 100 bootstrap replicates (parameters: -m GTRGAMMA -N 100). The resulting tree was visualized using iTOL (https://itol.embl.de) [81].

Supporting information

S1 Text. Fig A T6SS1 clusters share a similar synteny.

Comparison of T6SS1 clusters in the 31 V. coralliilyticus strains analyzed in this study. Colors denote homology between the encoded protein sequences. The strain name and RefSeq accession are denoted. Dashed, black vertical lines denote borders between separate contigs. Fig B. T6SS2 clusters share a similar synteny. Comparison of T6SS2 clusters in the 31 V. coralliilyticus strains analyzed in this study. Colors denote homology between the encoded protein sequences. The strain name and RefSeq accession are denoted. Dashed, black vertical lines denote borders between separate contigs. Fig C. Deletion of hcp1 or tssM2 inactivates T6SS1 or T6SS2, respectively. (A) Expression (cells) and secretion (media) of VgrG1 and Hcp2 from the indicated V. coralliilyticus strains grown for 4 h at 28°C in rich media (MLB). RNA polymerase sigma 70 (RNAp) was used as a loading and lysis control. Asterisks denote expected protein sizes. WT, wild-type. (B) The growth of the indicated V. coralliilyticus strains in MLB at 28°C measured as absorbance at 600 nm (OD600). Data are shown as the mean ± SD; n = 3. Results from a representative experiment out of at least 3 independent experiments are shown. The data underlying panel B of this figure can be found in S6 Data. Fig D. Vibrio coralliilyticus T6SS1 mediates interbacterial competition. (A, B) Viability counts (colony forming units; CFU) of V. alginolyticus 12G01 and V. campbellii ATCC 25920 prey strains before (0 h) and after (4 h) co-incubation with the indicated V. coralliilyticus OCN008 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; 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 3 independent experiments. The data underlying this figure can be found in S7 Data. Fig E. Non-structural proteins secreted by Vibrio coralliilyticus T6SS2 are encoded by orphan genes. Genomic neighborhoods of genes encoding representative T6SS effector proteins (CoVes) and their homologs (colored arrows). The strain names, the GenBank accession numbers, and protein accessions are denoted. Genes are denoted by arrows indicating the predicted direction of transcription. Gray rectangles denote regions of amino acid sequence homology; identity percentages are indicated. Fig F. CoVes are secreted in a T6SS2-dependent manner. Expression (cells) and secretion (media) of C-terminally FLAG-tagged CoVes expressed from arabinose-inducible plasmids in V. coralliilyticus strains, either wild-type (WT) or T6SS2− (ΔtssM2). CoVe1-8 were monitored in V. coralliilyticus BAA-450 and CoVe9 was monitored in V. coralliilyticus OCN008. V. coralliilyticus strains were grown in MLB supplemented with chloramphenicol and 0.01% (wt/vol) L-arabinose for 4 h at 28°C. Loading control (LC) is shown for total protein lysate. Results from a representative experiment out of at least 2 independent experiments are shown. Fig G. CoVes are expressed in E. coli. Expression of C-terminally FLAG-tagged CoVes from arabinose-inducible plasmids in E. coli strain DH5α (λ-pir). Loading control (LC) is shown for total protein lysate. Results from a representative experiment out of at least 2 independent experiments are shown. Table A. Bacteria and yeast strains used in this study. Table B. Plasmids used in this study. Table C. Primers used in this study.

(DOCX)

S1 Dataset OrthoANI analysis of V. coralliilyticus strains used in this study.

(XLSX)

S2 Dataset Analysis of V. coralliilyticus T6SS gene clusters.

(XLSX)

S3 Dataset Mass spectrometry results for V. coralliilyticus BAA-450 samples.

(XLSX)

S4 Dataset Mass spectrometry results for V. coralliilyticus OCN008 samples.

(XLSX)

S5 Dataset Mass spectrometry results for V. coralliilyticus OCN014 samples.

(XLSX)

S6 Dataset CoVe distribution in RefSeq V. coralliilyticus genomes.

(XLSX)

S1 Data Numerical values for Fig 3.

(XLSX)

S2 Data Numerical values for Fig 4.

(XLSX)

S3 Data Numerical values for Fig 5.

(XLSX)

S4 Data Numerical values for Fig 6.

(XLSX)

S5 Data Tree data for Fig 8.

(TXT)

S6 Data Numerical values for panel B of Fig C in S1 Text.

(XLSX)

S7 Data Numerical values for Fig D in S1 Text.

(XLSX)

S1 Raw Images Uncropped and minimally adjusted images supporting all blot results included in the article.

(PDF)

We thank members of the Salomon, van Kessel, and Ushijima groups for valuable discussions, and Katarzyna Kanarek for preparing the pKara1 plasmid. We also thank the Smoler Proteomics Center at the Technion for performing and analyzing the mass spectrometry data.

Abbreviations

BMDM bone marrow-derived macrophage

CDS coding sequences

CFU colony-forming unit

CoVe Coralliilyticus virulence effector

CNF cytotoxic necrotizing factor

ECL enhanced chemiluminescence

eGFP enhanced GFP

FDR false discovery rate

GASW glycerol artificial seawater

LB lysogeny broth

LFQ Label-free quantification

MCS multiple cloning site

MLB marine lysogeny broth

MOI multiplicity of infection

PI propidium iodide

T6SS type VI secretion system

WT wild-type

10.1371/journal.pbio.3002734.r001
Decision Letter 0
Vazquez Hernandez Melissa Senior Editor
© 2024 Melissa Vazquez Hernandez
2024
Melissa Vazquez Hernandez
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
2 Apr 2024

Dear Dr Salomon,

Thank you for submitting your manuscript entitled "A T6SS in the coral pathogen Vibrio coralliilyticus secretes an arsenal of anti-eukaryotic effectors and contributes to virulence" for consideration as a Research Article by PLOS Biology. I would like to apologize for the long time it took me to send you an initial decision.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Apr 04 2024 11:59PM.

If your manuscript has been previously peer-reviewed at another journal, PLOS Biology is willing to work with those reviews in order to avoid re-starting the process. Submission of the previous reviews is entirely optional and our ability to use them effectively will depend on the willingness of the previous journal to confirm the content of the reports and share the reviewer identities. Please note that we reserve the right to invite additional reviewers if we consider that additional/independent reviewers are needed, although we aim to avoid this as far as possible. In our experience, working with previous reviews does save time.

If you would like us to consider previous reviewer reports, please edit your cover letter to let us know and include the name of the journal where the work was previously considered and the manuscript ID it was given. In addition, please upload a response to the reviews as a 'Prior Peer Review' file type, which should include the reports in full and a point-by-point reply detailing how you have or plan to address the reviewers' concerns.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Melissa

Melissa Vazquez-Hernandez, Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org

10.1371/journal.pbio.3002734.r002
Decision Letter 1
Vazquez Hernandez Melissa Senior Editor
© 2024 Melissa Vazquez Hernandez
2024
Melissa Vazquez Hernandez
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
21 May 2024

Dear Dr Salomon,

Thank you for your patience while your manuscript "A T6SS in the coral pathogen Vibrio coralliilyticus secretes an arsenal of anti-eukaryotic effectors and contributes to virulence" went through peer-review at PLOS Biology. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers, with reviewers Xiaoxue Wang (R1) and Frederique Le Roux (R2) identifying themselves. I would like to apologize for the delay in giving you a decision.

In light of the reviews, which you will find at the end of this email, we are pleased to offer you the opportunity to address the comments from the reviewers in a revision that we anticipate should not take you very long. As you will see in the reports, all reviewers are positive about the relevance of the work. Reviewer #1 and #2 suggest to evaluate the pathogenicity of effector mutants, and Reviewer also #1 suggests to perform the competition experiment in a more representative environment. However, while we believe the experimental requests would strengthen the conclusions, they are not required for publication in our view. We leave it to your discretion if you want to include these or not. All other reviewer concerns and suggestions should be addressed for further consideration.

We will then assess your revised manuscript and your response to the reviewers' comments with our Academic Editor aiming to avoid further rounds of peer-review, although might need to consult with the reviewers, depending on the nature of the revisions.

We expect to receive your revised manuscript within 1 month. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we withdraw the manuscript.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point-by-point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Revised Article with Changes Highlighted " file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org

----------------------------------------------------------------

REVIEWERS' COMMENTS:

----------------------------------------------------------------

Reviewer #1:

The manuscript by Shir Mass et al., titled "A T6SS in the coral pathogen Vibrio coralliilyticus secretes an arsenal 2 of anti-eukaryotic effectors and contributes to virulence," analyzed the T6SS in Vibrio coralliilyticus and identified two omnipresent T6SS systems, T6SS1 and T6SS2. T6SS1 is involved in interbacterial competition, while T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of Artemia salina. Through comparative proteomics, they identified the effectors of T6SS1 and T6SS2 in three Vcor strains, including nine novel effectors secreted by T6SS2. The results of this study are interesting and informative. Overall, it is a well-organized and well-written manuscript with rigorous logic.

There is a bit room for improvement and I have only two minor comments:

1. The only downside is the lack of coral infection assay mediated by T6SS2 effectors.

On line 122, the authors performed comparative proteomics analyses to reveal the Vcor T6SS secretomes and identify the effectors. If possible, the mutant strains by deleting genes encoding effectors like toxic proteins could be constructed and used to test the pathogenicity for Artemia salina.

2. The authors competed Vcor strains against a sensitive a V. natriegens prey strain on rich media. It will be better if the selected vibrio strain is more representative or isolated in the natural environment, such as coral tissue, gastric cavity of shellfish larvae.

BTW, on line 164, "a sensitive a V. natriegens" into "a sensitive V. natriegens".

----------------------------------------------------------------

Reviewer #2:

This study presents compelling evidence that two Type VI Secretion Systems (T6SS) are conserved in Vibrio coralliilyticus, a pathogen affecting corals and oysters. Both T6SS systems are activated at ecologically relevant temperatures (28°C), yet they differ in their secretion profiles: T6SS1 targets prokaryotes, while T6SS2 secretes anti-eukaryotic effectors. Notably, T6SS2 induces mortality in artemia, serving as a model system for pathogenicity assessment. Through comparative proteomics, the authors identified nine novel putative toxins that specifically target eukaryotic cells, shedding light on the diversity of T6SS effectors.

Overall, the data presented are convincing and innovative, supported by a well-designed experimental approach. This study underscores the vast potential for uncovering new insights into T6SS dynamics using non-human Vibrio pathogens.

I only have minor comments

Introduction

The assertion regarding the spread of vibrios to new regions correlating with rising ocean temperatures might benefit from rephrasing to avoid potential misinterpretation. Indeed I disagree with this statement. Vibrios encompass a wide range of species inhabiting diverse environments, including psychrophiles. There is a tendency for publications to conflate human pathogens like V. cholerae and V. parahaemolyticus with the broader category of vibrios, because research historically focused heavily on V. cholerae.

Additionally, please use "V. coralliilyticus" instead of "Vcor" everywhere

References to Rubio et al. (PNAS, 2019) and works from the Blokesch lab, particularly Drebes Dörr et al. (Environmental Microbiology, 2020), should be included for comprehensive coverage.

Results:

Consider incorporating a figure depicting the core genome phylogeny of the 31 V. coralliilyticus strains and the synteny of T6SS1 and T6SS2 to facilitate data interpretation.

Clarify the term "Marine LB" (Line 134) for improved understanding.

Regarding the comparison of T6SS activity between rich and poor media at 28°C (Line 142), adjust the statement to reflect the relative protein abundance differences more accurately (clearly less proteins in poor media).

For Figures 4B, C, D, provide clarification on the statistical significance of the results, particularly regarding the differences between T6SS1 and T6SS2 mutants.

It appears that for one strain T6SS1 has an effect (linked to MIX domain-containing effectors?)

In Figure 8, consider presenting a core genome phylogeny and include bootstrap values for clarity.

Further clarification is needed regarding the rationale behind selecting strains for phylogeny in Figure 8 and the presence of homologs of CoVe2 and CoVe8 in all V. coralliilyticus genomes. Do they mean paralog/gene duplication?

Consideration could be given to testing the deletion of candidate toxins in cellular assays, such as BMDM, for a more comprehensive understanding of their role in pathogenicity. If they tried but did not get an effect, what does it mean?

Discussion

Reevaluate the necessity of certain paragraphs, such as lines 355-364, for enhanced coherence and relevance.

Line 380: Ensure clarity in statements regarding the nature of Artemia infection assays. I don't believe V. coralliilyticus kills artemia externally, but rather after being ingested. Consequently, the bacteria are not exposed to ocean salt but instead to the stomach environment, which may not be nutrient-poor.

Lines 387-389. What is meant here? Is it suggesting that V. coralliilyticus could be a human pathogen? This assertion seems somewhat speculative and should not be the concluding statement. However, I find it intriguing that they conducted cellular assays with mouse cells, considering that Vibrio species from marine environments (non-human pathogen) typically struggle in salt-free media.

Provide a stronger conclusion that encapsulates the main findings and implications of the study.

----------------------------------------------------------------

Reviewer #3:

The authors report studies on the role of the Type 6 Secretion Systems (T6SSs) of the bacterial organism Vibrio coralliilyticus (Vcor) in assays for anrti-bacterial, anti-eukaryotic phagocytic cells, and anti-higher organisms (Artemia salina or brine shrimp). Because Vcor is a significant pathogen of both coral and shellfish, this study is potentially of high importance to understanding and potentially controlling this aquatic bacterial pathogen.

The investigators performed studies to address whether the expression of two conserved ("omipresent') T6SSs of Vcor were regulated by growth temperature and found that indeed these were induced by elevated temperatures --- conditions that make Vcor particularly pathogenic for the model organism Artemia salina. This result is important because previous studies have proposed that elevated ocean temperatures modulate the virulence of this organism particularly for coral in the context of bleaching disease.

The investigators go on to convincingly show that the T6SS1 has a major role as an antibacterial weapon using standard co-incubation on agar assays. They then show that the T6SS2 can elicit toxicity in both shrimp and in bone marrow derived macrophages from mice. These anti-eukaryotic effects of the T6SS are relatively rare in the field and thus these are important new results. The toxicity observed also correlated with loss of viability of the shrimp in their aquatic animal model. Using state-of-the-art proteomics the authors also determined the array of proteins that were likely secreted by the T6SS machines in Vcor. These included the usual structural components like Hcp, VgrG, and PAAR proteins but also a variety of likely effector proteins based on their homology to T6SS effector in other bacterial systems. Several of these proteins also did not display homology to known effectors and thus may indeed be novel toxins for prokaryotic or eukaryotic cells.

I found the manuscript well-written and now reasonable to accept for publication in PLOS Biology. It addresses an important problem in microbiology -- how rising sea temperatures might affect the fitness and virulence of aquatic bacterial that in turn can then cause major economic and ecological damage globally.

10.1371/journal.pbio.3002734.r003
Author response to Decision Letter 1
Submission Version2
14 Jun 2024

Attachment Submitted filename: PBP.docx

10.1371/journal.pbio.3002734.r004
Decision Letter 2
Vazquez Hernandez Melissa Senior Editor
© 2024 Melissa Vazquez Hernandez
2024
Melissa Vazquez Hernandez
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
27 Jun 2024

Dear Dr Salomon,

Thank you for your patience while we considered your revised manuscript "A T6SS in the coral pathogen Vibrio coralliilyticus secretes an arsenal of anti-eukaryotic effectors and contributes to virulence" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor.

Based on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication. Please also make sure to address the following data and other policy-related requests.

a) We routinely suggest changes to titles to ensure maximum accessibility for a broad, non-specialist readership, and to ensure they reflect the contents of the paper. In this case, we would suggest a minor edit to the title, as follows. Please ensure you change both the manuscript file and the online submission system, as they need to match for final acceptance.

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

b) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Please supply the numerical values either in the a supplementary file or as a permanent DOI’d deposition for the following figures:

Figure 3ABC, 4ABCD, 5, 6, S3B, S4

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

c) Please cite the location of the data clearly in all relevant main and supplementary Figure legends, e.g. “The data underlying this Figure can be found in S1 Data” or “The data underlying this Figure can be found in https://doi.org/10.5281/zenodo.XXXXX”

d) Please provide the tree file for Figure 8

e) We thank you for providing the uncropped and minimally adjusted images supporting all blot and gel results reported. However please note that the labelling fin the raw images files, seems to be wrong in regard to the supplementary figures. Raw gels for S3A, S6, and S7, seemed to be now labelled as S1A, S3, and S4.

f) Please ensure that your Data Statement in the submission system accurately describes where your data can be found and is in final format, as it will be published as written there.

g) Per journal policy, if you have generated any custom code during the curse of this investigation, please make it available without restrictions upon publication. Please ensure that the code is sufficiently well documented and reusable, and that your Data Statement in the Editorial Manager submission system accurately describes where your code can be found.

Please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

As you address these items, please take this last chance to review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

mvazquezhernandez@plos.org

PLOS Biology

10.1371/journal.pbio.3002734.r005
Author response to Decision Letter 2
Submission Version3
28 Jun 2024

Attachment Submitted filename: PBP.docx

10.1371/journal.pbio.3002734.r006
Decision Letter 3
Vazquez Hernandez Melissa Senior Editor
© 2024 Melissa Vazquez Hernandez
2024
Melissa Vazquez Hernandez
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version3
3 Jul 2024

Dear Dr Salomon,

Thank you for the submission of your revised Research Article "The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, [**AE Name**], I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Melissa

Melissa Vazquez Hernandez, Ph.D., Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org
==== Refs
References

1 Horseman MA , Bray R , Lujan-Francis B , Matthew E . Infections Caused by Vibrionaceae. Infect Dis Clin Pract. 2013;21 :222–232. doi: 10.1097/IPC.0b013e3182826328
2 Baker-Austin C , Oliver JD , Alam M , Ali A , Waldor MK , Qadri F , et al . Vibrio spp. infections. Nat Rev Dis Prim. 2018;4 :1–19. doi: 10.1038/s41572-018-0005-8 29930242
3 Martinez-Urtaza J , Baker-Austin C , Jones JL , Newton AE , Gonzalez-Aviles GD , DePaola A . Spread of Pacific Northwest Vibrio parahaemolyticus Strain. N Engl J Med. 2013;369 :1573–1574. doi: 10.1056/NEJMc1305535 24131194
4 Le Roux F , Wegner KM , Baker-Austin C , Vezzulli L , Osorio CR , Amaro C , et al . The emergence of Vibrio pathogens in Europe: Ecology, evolution and pathogenesis (Paris, 11–12 March 2015). Front Microbiol. 2015;6 :1–8. doi: 10.3389/fmicb.2015.00830 25653648
5 Vezzulli L , Grande C , Reid PC , Hélaouët P , Edwards M , Höfle MG , et al . Climate influence on Vibrio and associated human diseases during the past half-century in the coastal North Atlantic. Proc Natl Acad Sci U S A. 2016;113 :E5062–E5071. doi: 10.1073/pnas.1609157113 27503882
6 Newton A , Kendall M , Vugia DJ , Henao OL , Mahon BE . Increasing Rates of Vibriosis in the United States, 1996–2010: Review of Surveillance Data From 2 Systems. Clin Infect Dis. 2012;54 :S391–S395. doi: 10.1093/cid/cis243 22572659
7 Burke S , Pottier P , Lagisz M , Macartney EL , Ainsworth T , Drobniak SM , et al . The impact of rising temperatures on the prevalence of coral diseases and its predictability: A global meta-analysis. Ecol Lett. 2023;26 :1466–1481. doi: 10.1111/ele.14266 37278985
8 Arboleda M , Reichardt W . Epizoic communities of prokaryotes on healthy and diseased scleractinian corals in Lingayen Gulf, Philippines. Microb Ecol. 2009;57 :117–128. doi: 10.1007/s00248-008-9400-0 18535850
9 Moriarty T , Leggat W , Huggett MJ , Ainsworth TD . Coral Disease Causes, Consequences, and Risk within Coral Restoration. Trends Microbiol. 2020:793–807. doi: 10.1016/j.tim.2020.06.002 32739101
10 Elliff CI , Silva IR . Coral reefs as the first line of defense: Shoreline protection in face of climate change. Marine Environ Res. 2017:148–154. doi: 10.1016/j.marenvres.2017.03.007 28366280
11 Mera H , Bourne DG . Disentangling causation: complex roles of coral-associated microorganisms in disease. Environ Microbiol. 2018:431–449. doi: 10.1111/1462-2920.13958 29027742
12 Bender-Champ D , Diaz-Pulido G , Dove S . Effects of elevated nutrients and CO2 emission scenarios on three coral reef macroalgae. Harmful Algae. 2017;65 :40–51. doi: 10.1016/j.hal.2017.04.004 28526118
13 Vanwonterghem I , Webster NS . Coral Reef Microorganisms in a Changing Climate. iScience. 2020. p. 100972. doi: 10.1016/j.isci.2020.100972 32208346
14 Tout J , Siboni N , Messer LF , Garren M , Stocker R , Webster NS , et al . Increased seawater temperature increases the abundance and alters the structure of natural Vibrio populations associated with the coral Pocillopora damicornis. Front Microbiol. 2015;6 :432. doi: 10.3389/fmicb.2015.00432 26042096
15 Ben-Haim Y , Zicherman-Keren M , Rosenberg E . Temperature-regulated bleaching and lysis of the coral Pocillopora damicornis by the novel pathogen Vibrio coralliilyticus. Appl Environ Microbiol. 2003;69 :4236–4242. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12839805. doi: 10.1128/AEM.69.7.4236-4242.2003 12839805
16 Ushijima B , Videau P , Burger AH , Shore-Maggio A , Runyon CM , Sudek M , et al . Vibrio coralliilyticus strain OCN008 is an etiological agent of acute montipora white syndrome. Appl Environ Microbiol. 2014;80 :2102–2109. doi: 10.1128/AEM.03463-13 24463971
17 Richards GP , Watson MA , Needleman DS , Church KM , Häse CC . Mortalities of Eastern And Pacific oyster larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii. Appl Environ Microbiol. 2015;81 :292–297. doi: 10.1128/AEM.02930-14 25344234
18 Ushijima B , Richards GP , Watson MA , Schubiger CB , Häse CC . Factors affecting infection of corals and larval oysters by Vibrio coralliilyticus. PLoS ONE. 2018:13. doi: 10.1371/journal.pone.0199475 29920567
19 Kimes NE , Grim CJ , Johnson WR , Hasan NA , Tall BD , Kothary MH , et al . Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus. ISME J. 2012;6 :835–846. doi: 10.1038/ismej.2011.154 22158392
20 Childers BM , Klose KE . Regulation of virulence in Vibrio cholerae: The ToxR regulon. Future Microbiol. 2007:335–344. doi: 10.2217/17460913.2.3.335 17661707
21 Ushijima B , Videau P , Poscablo D , Stengel JW , Beurmann S , Burger AH , et al . Mutation of the toxR or mshA genes from Vibrio coralliilyticus strain OCN014 reduces infection of the coral Acropora cytherea. Environ Microbiol. 2016;18 :4055–4067. doi: 10.1111/1462-2920.13428 27348808
22 Dar Y , Salomon D , Bosis E . The antibacterial and anti-eukaryotic Type VI secretion system MIX-effector repertoire in Vibrionaceae. Mar Drugs. 2018;16 :433. doi: 10.3390/md16110433 30400344
23 Pukatzki S , Ma AT , Sturtevant D , Krastins B , Sarracino D , Nelson WC , et al . Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A. 2006;103 :1528–1533. doi: 10.1073/pnas.0510322103 16432199
24 Salomon D , Gonzalez H , Updegraff BL , Orth K . Vibrio parahaemolyticus Type VI secretion system 1 Is activated in marine conditions to target bacteria, and is differentially regulated from system 2. PLoS ONE. 2013;8 :e61086. doi: 10.1371/journal.pone.0061086 23613791
25 Tchelet D , Keppel K , Bosis E , Salomon D . Vibrio parahaemolyticus T6SS2 effector repertoires. Gut Microbes. 2023;15 :2178795. doi: 10.1080/19490976.2023.2178795 36803660
26 Salomon D , Klimko JA , Trudgian DC , Kinch LN , Grishin NV , Mirzaei H , et al . Type VI secretion system toxins horizontally shared between marine bacteria. PLoS Pathog. 2015;11 :e1005128. doi: 10.1371/journal.ppat.1005128 26305100
27 Speare L , Cecere AG , Guckes KR , Smith S , Wollenberg MS , Mandel MJ , et al . Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proc Natl Acad Sci U S A. 2018;115 :E8528–E8537. doi: 10.1073/pnas.1808302115 30127013
28 Ray A , Schwartz N , Souza Santos M , Zhang J , Orth K , Salomon D , et al . Type VI secretion system MIX-effectors carry both antibacterial and anti-eukaryotic activities. EMBO Rep. 2017;18 :e201744226. doi: 10.15252/embr.201744226 28912123
29 Piel D , Bruto M , James A , Labreuche Y , Lambert C , Janicot A , et al . Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells. Environ Microbiol. 2020;22 :4198–4211. doi: 10.1111/1462-2920.14776 31390475
30 Wang J , Brodmann M , Basler M . Assembly and subcellular localization of bacterial type VI secretion systems. Annu Rev Microbiol. 2019;73 :621–638. doi: 10.1146/annurev-micro-020518-115420 31226022
31 Jana B , Salomon D . Type VI secretion system: a modular toolkit for bacterial dominance. Future Microbiol. 2019;14 :fmb-2019-0194. doi: 10.2217/fmb-2019-0194 31718312
32 Hernandez RE , Gallegos-Monterrosa R , Coulthurst SJ . Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness. Cell Microbiol. 2020:e13241. doi: 10.1111/cmi.13241 32592518
33 Allsopp LP , Bernal P . Killing in the name of: T6SS structure and effector diversity. Microbiology. 2023;169 :001367. doi: 10.1099/mic.0.001367 37490402
34 Basler M , Pilhofer M , Henderson GP , Jensen GJ , Mekalanos JJ . Type VI secretion requires a dynamic contractile phage tail-like structure. Nature. 2012;483 :182–186. doi: 10.1038/nature10846 22367545
35 Hachani A , Wood TE , Filloux A . Type VI secretion and anti-host effectors. Curr Opin Microbiol. 2016;29 :81–93. doi: 10.1016/j.mib.2015.11.006 26722980
36 Monjarás Feria J , Valvano MA . An Overview of Anti-Eukaryotic T6SS Effectors. Frontiers in Cellular and Infection. Microbiology. 2020:584751. doi: 10.3389/fcimb.2020.584751 33194822
37 Huang Y , Du P , Zhao M , Liu W , Du Y , Diao B , et al . Functional characterization and conditional regulation of the type VI secretion system in Vibrio fluvialis. Front Microbiol. 2017;8 :1–15. doi: 10.3389/fmicb.2017.00528 28197127
38 MacIntyre DL , Miyata ST , Kitaoka M , Pukatzki S . The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci U S A. 2010;107 :19520–19524. doi: 10.1073/pnas.1012931107 20974937
39 Church SR , Lux T , Baker-Austin C , Buddington SP , Michell SL . Vibrio vulnificus type 6 secretion system 1 contains anti-bacterial properties. PLoS ONE. 2016;11 :1–17. doi: 10.1371/journal.pone.0165500 27798649
40 Cohen H , Baram N , Fridman CM , Edry-Botzer L , Salomon D , Gerlic M . Post-phagocytosis activation of NLRP3 inflammasome by two novel T6SS effectors. Elife. 2022;11 :e82766. doi: 10.7554/eLife.82766 36155655
41 Cohen H , Fridman CM , Gerlic M , Salomon D . A Vibrio T6SS-Mediated Lethality in an Aquatic Animal Model. Cascales E , editor. Microbiol Spectr. 2023;11. doi: 10.1128/spectrum.01093-23 37310285
42 Kanarek K , Fridman CM , Bosis E , Salomon D . The RIX domain defines a class of polymorphic T6SS effectors and secreted adaptors. Nat Commun. 2023;14 :4983. doi: 10.1038/s41467-023-40659-2 37591831
43 Bruto M , James A , Petton B , Labreuche Y , Chenivesse S , Alunno-Bruscia M , et al . Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition. ISME J. 2017;11 :1043–1052. doi: 10.1038/ismej.2016.162 27922600
44 Drebes Dörr NC , Blokesch M . Interbacterial competition and anti-predatory behaviour of environmental Vibrio cholerae strains. Environ Microbiol. 2020;22 :4485–4504. doi: 10.1111/1462-2920.15224 32885535
45 Rubio T , Oyanedel D , Labreuche Y , Toulza E , Luo X , Bruto M , et al . Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections. Proc Natl Acad Sci U S A. 2019;116 :14238–14247. doi: 10.1073/pnas.1905747116 31221761
46 Guillemette R , Ushijima B , Jalan M , Häse CC , Azam F . Insight into the resilience and susceptibility of marine bacteria to T6SS attack by Vibrio cholerae and Vibrio coralliilyticus. PLoS ONE. 2020;15 :e0227864. doi: 10.1371/journal.pone.0227864 31990915
47 Salomon D , Kinch LN , Trudgian DC , Guo X , Klimko JA , Grishin NV , et al . Marker for type VI secretion system effectors. Proc Natl Acad Sci U S A. 2014;111 :9271–9276. doi: 10.1073/pnas.1406110111 24927539
48 Dar Y , Jana B , Bosis E , Salomon D . A binary effector module secreted by a type VI secretion system. EMBO Rep. 2022;23 :e53981. doi: 10.15252/embr.202153981 34752000
49 Ronholm J , Petronella N , Chew Leung C , Pightling AW , Banerjeea SK . Genomic features of environmental and clinical Vibrio parahaemolyticus isolates lacking recognized virulence factors are dissimilar. Appl Environ Microbiol. 2016;82 :1102–1113. doi: 10.1128/AEM.03465-15 26637607
50 Li P , Kinch LN , Ray A , Dalia AB , Cong Q , Nunan LM , et al . Acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus strains maintain an antibacterial type VI secretion system with versatile effector repertoires. Appl Environ Microbiol. 2017;83 :e00737–e00717. doi: 10.1128/AEM.00737-17 28432099
51 Ben-Haim Y , Thompson FL , Thompson CC , Cnockaert MC , Hoste B , Swings J , et al . Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis. Int J Syst Evol Microbiol. 2003;53 :309–315. doi: 10.1099/ijs.0.02402-0 12656189
52 Ushijima B , Videau P , Poscablo D , Vine V , Salcedo M , Aeby G , et al . Complete genome sequence of Vibrio coralliilyticus strain OCN014, isolated from a diseased coral at Palmyra Atoll. Genome Announc. 2014;2 :e01318–e01314. doi: 10.1128/genomeA.01318-14 25523774
53 Alves N , Maia Neto OS , Silva BSO , de Moura RL , Francini-Filho RB , et al . Diversity and pathogenic potential of vibrios isolated from Abrolhos Bank corals. Environ Microbiol Rep. 2010;2 :90–95. doi: 10.1111/j.1758-2229.2009.00101.x 23766002
54 Neu AK , Månsson M , Gram L , Prol-García MJ . Toxicity of bioactive and probiotic marine bacteria and their secondary metabolites in artemia sp. and caenorhabditis elegans as eukaryotic model organisms. Appl Environ Microbiol. 2014;80 :146–153. doi: 10.1128/AEM.02717-13 24141121
55 Austin B , Austin D , Sutherland R , Thompson F , Swings J . Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii. Environ Microbiol. 2005;7 :1488–1495. doi: 10.1111/j.1462-2920.2005.00847.x 16104871
56 Jana B , Keppel K , Fridman CM , Bosis E , Salomon D . Multiple T6SSs, mobile auxiliary modules, and effectors revealed in a systematic analysis of the Vibrio parahaemolyticus pan-genome. Bordenstein S , editor. mSystems. 2022;e00723–22. doi: 10.1128/msystems.00723-22 36226968
57 Iriarte M , Cornelis GR . YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells. Mol Microbiol. 1998;29 :915–929. doi: 10.1046/j.1365-2958.1998.00992.x 9723929
58 Teufel F , Almagro Armenteros JJ , Johansen AR , Gíslason MH , Pihl SI , Tsirigos KD , et al . SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol. 2022;40 :1023–1025. doi: 10.1038/s41587-021-01156-3 34980915
59 Deng Q , Barbieri JT . Molecular mechanisms of the cytotoxicity of ADP-ribosylating toxins. Annu Rev Microbiol. 2008:271–288. doi: 10.1146/annurev.micro.62.081307.162848 18785839
60 Knust Z , Schmidt G . Cytotoxic necrotizing factors (CNFs)-a growing toxin family. Toxins. 2010:116–127. doi: 10.3390/toxins2010116 22069550
61 Ahmad S , Wang B , Walker MD , Tran HKR , Stogios PJ , Savchenko A , et al . An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp. Nature. 2019;575 :674–678. doi: 10.1038/s41586-019-1735-9 31695193
62 Siggers KA , Lesser CF . The Yeast Saccharomyces cerevisiae: A Versatile Model System for the Identification and Characterization of Bacterial Virulence Proteins. Cell Host Microbe. 2008;4 :8–15. doi: 10.1016/j.chom.2008.06.004 18621006
63 Popa C , Coll NS , Valls M , Sessa G . Yeast as a Heterologous Model System to Uncover Type III Effector Function. Bliska JB , editor. PLoS Pathog. 2016;12 :e1005360. doi: 10.1371/journal.ppat.1005360 26914889
64 Robinson LA , Collins ACZ , Murphy RA , Davies JC , Allsopp LP . Diversity and prevalence of type VI secretion system effectors in clinical Pseudomonas aeruginosa isolates. Front Microbiol. 2023;13 :1042505. doi: 10.3389/fmicb.2022.1042505 36687572
65 Unterweger D , Miyata ST , Bachmann V , Brooks TM , Mullins T , Kostiuk B , et al . The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition. Nat Commun. 2014;5 :3549. doi: 10.1038/ncomms4549 24686479
66 Dunn AK , Millikan DS , Adin DM , Bose JL , Stabb EV . New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl Environ Microbiol. 2006;72 :802–810. doi: 10.1128/AEM.72.1.802–810.2006 16391121
67 Gibson DG , Young L , Chuang RY , Venter JC , Hutchison CA , Smith HO . Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6 :343–345. doi: 10.1038/nmeth.1318 19363495
68 Salomon D , Sessa G . Identification of growth inhibition phenotypes induced by expression of bacterial type III effectors in yeast. J Vis Exp. 2010:4–7. doi: 10.3791/1865 20354502
69 Bensadoun A , Weinstein D . Assay of proteins in the presence of interfering materials. Anal Biochem. 1976;70 :241–250. doi: 10.1016/s0003-2697(76)80064-4 1259145
70 Cox J , Hein MY , Luber CA , Paron I , Nagaraj N , Mann M . Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 2014;13 :2513–2526. doi: 10.1074/mcp.M113.031591 24942700
71 Tyanova S , Temu T , Sinitcyn P , Carlson A , Hein MY , Geiger T , et al . The Perseus computational platform for comprehensive analysis of (prote)omics data. Nature Methods. 2016:731–740. doi: 10.1038/nmeth.3901 27348712
72 Perez-Riverol Y , Bai J , Bandla C , García-Seisdedos D , Hewapathirana S , Kamatchinathan S , et al . The PRIDE database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50 :D543–D552. doi: 10.1093/nar/gkab1038 34723319
73 Chung HS , Raetz CRH . Interchangeable domains in the Kdo transferases of escherichia coli and haemophilus influenzae. Biochemistry. 2010;49 :4126–4137. doi: 10.1021/bi100343e 20394418
74 Erlich Z , Shlomovitz I , Edry-Botzer L , Cohen H , Frank D , Wang H , et al . Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model. Nat Immunol. 2019:397–406. doi: 10.1038/s41590-019-0313-5 30742078
75 Lee I , Ouk Kim Y , Park S-C , Chun J . OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 2016;66 :1100–1103. doi: 10.1099/ijsem.0.000760 26585518
76 Fridman CM , Keppel K , Gerlic M , Bosis E , Salomon D . A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors. Nat Commun. 2020;11 :1085. doi: 10.1038/s41467-020-14951-4 32109231
77 Gilchrist CLM , Chooi YH . Clinker & clustermap.js: Automatic generation of gene cluster comparison figures. Bioinformatics. 2021;37 :2473–2475. doi: 10.1093/bioinformatics/btab007 33459763
78 Seppey M , Manni M , Zdobnov EM . BUSCO: Assessing genome assembly and annotation completeness. Methods Mol Biol. 2019:227–245. doi: 10.1007/978-1-4939-9173-0_14 31020564
79 Edgar RC . MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5 :113. doi: 10.1186/1471-2105-5-113 15318951
80 Stamatakis A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30 :1312–1313. doi: 10.1093/bioinformatics/btu033 24451623
81 Letunic I , Bork P . Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res. 2024;2024 :1–5. doi: 10.1093/NAR/GKAE268 38613393
