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Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria.

Salomon D, Klimko JA, Trudgian DC, Kinch LN, Grishin NV, Mirzaei H, Orth K - PLoS Pathog. (2015)

Bottom Line: Using comparative proteomics and genetics, we identified their effector repertoires.We also showed that the T6SS2 secretes at least three antibacterial effectors.We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are "orphan" effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness.

No MeSH data available.


Va16922/Va16927, Va18287/Va18282, and Va03175/Va03170/Va03180 are VaT6SS2 effector/immunity pairs.(A) Schematic representation of the VaT6SS2 gene cluster and effector/immunity pairs. V12G01 locus numbers listed above. Effectors in red, Immunity in green, and tail tube components in orange. (B-D) Viability counts of prey strains containing an empty plasmid or a plasmid for the arabinose-inducible expression of the immunity protein before (0h) and after (4h) co-culture with the indicated attacker strains. Effector/immunity pairs tested were: (B) Va16922/Va16927, (C) Va18287/Va18282, and (D) Va03175/Va03170/Va03180. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).
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ppat.1005128.g002: Va16922/Va16927, Va18287/Va18282, and Va03175/Va03170/Va03180 are VaT6SS2 effector/immunity pairs.(A) Schematic representation of the VaT6SS2 gene cluster and effector/immunity pairs. V12G01 locus numbers listed above. Effectors in red, Immunity in green, and tail tube components in orange. (B-D) Viability counts of prey strains containing an empty plasmid or a plasmid for the arabinose-inducible expression of the immunity protein before (0h) and after (4h) co-culture with the indicated attacker strains. Effector/immunity pairs tested were: (B) Va16922/Va16927, (C) Va18287/Va18282, and (D) Va03175/Va03170/Va03180. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).

Mentions: We predicted that three of the VaT6SS2 secreted proteins: Va16922, Va18287, and Va03175, were antibacterial effectors as they possess predicted nuclease (Va16922 and Va18287) or pore-forming colicin-like (Va03175) domains (according to HHPred analysis [29]) that can mediate antibacterial toxicity. Moreover, the genes encoding for these three proteins were immediately upstream of small open reading frames (ORFs) that could encode for their cognate immunity proteins (Va16927, Va18282, and Va03180, respectively). These three putative effector/immunity pairs were encoded outside of the VaT6SS2 gene cluster (Fig 2A). To test whether Va16922/7, Va18287/2, and Va03175/80 are VaT6SS2 effector/immunity pairs, we monitored the ability of a V. alginolyticus wild-type strain, which is immune against self-intoxication, to kill strains with deletions in the putative effector/immunity gene pairs. Indeed, the wild-type strain was able to kill strains with deletions in va16922-7 and va18287-2 when co-cultured under VaT6SS2 inducing conditions (Fig 2B and 2C), indicating that deletion of these bicistronic units resulted in loss of immunity against self-intoxication. However, a strain deleted for va03175-80 was still immune against self-intoxication (Fig 2D), suggesting that either Va03175/80 are not an effector/immunity pair, or that there is an additional immunity gene. Using Va03180 as template, we performed a BLAST search to look for possible redundant immunity proteins in V. alginolyticus 12G01. We identified Va03170, encoded by the gene immediately upstream of the putative effector Va03175, as a homolog of Va03180 (65% identity). Therefore, we generated a strain deleted for the putative effector and the two homologous putative immunity genes, Δva03170-80. As predicted, the wild-type strain was able to kill the Δva03170-80 strain when co-cultured under VaT6SS2 inducing conditions (Fig 2D), indicating that va03170 and va03180 encode for redundant immunity proteins against Va03175-medited toxicity. In all cases, inactivation of VaT6SS2 by deletion of hcp2, or deletion of the effector/immunity pair in the attacking strain, resulted in loss of self-intoxication indicating that the toxic effectors were delivered by VaT6SS2 and encoded within these bicistronic units. Moreover, exogenous expression of the putative immunity proteins from a plasmid in the prey strains deleted for the effector/immunity pairs restored immunity against self-intoxication (Fig 2). Importantly, expression of either Va03170 or Va03180 from a plasmid restored immunity against self-intoxication in the Δva03170-80 strain indicating they are indeed redundant immunity proteins against Va03175-mediated toxicity (Fig 2D and S2 Fig). Taken together, these results demonstrate that Va16922/7, Va18287/2, and Va03175/80/70, are effector/immunity pairs of VaT6SS2.


Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria.

Salomon D, Klimko JA, Trudgian DC, Kinch LN, Grishin NV, Mirzaei H, Orth K - PLoS Pathog. (2015)

Va16922/Va16927, Va18287/Va18282, and Va03175/Va03170/Va03180 are VaT6SS2 effector/immunity pairs.(A) Schematic representation of the VaT6SS2 gene cluster and effector/immunity pairs. V12G01 locus numbers listed above. Effectors in red, Immunity in green, and tail tube components in orange. (B-D) Viability counts of prey strains containing an empty plasmid or a plasmid for the arabinose-inducible expression of the immunity protein before (0h) and after (4h) co-culture with the indicated attacker strains. Effector/immunity pairs tested were: (B) Va16922/Va16927, (C) Va18287/Va18282, and (D) Va03175/Va03170/Va03180. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).
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Related In: Results  -  Collection

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ppat.1005128.g002: Va16922/Va16927, Va18287/Va18282, and Va03175/Va03170/Va03180 are VaT6SS2 effector/immunity pairs.(A) Schematic representation of the VaT6SS2 gene cluster and effector/immunity pairs. V12G01 locus numbers listed above. Effectors in red, Immunity in green, and tail tube components in orange. (B-D) Viability counts of prey strains containing an empty plasmid or a plasmid for the arabinose-inducible expression of the immunity protein before (0h) and after (4h) co-culture with the indicated attacker strains. Effector/immunity pairs tested were: (B) Va16922/Va16927, (C) Va18287/Va18282, and (D) Va03175/Va03170/Va03180. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).
Mentions: We predicted that three of the VaT6SS2 secreted proteins: Va16922, Va18287, and Va03175, were antibacterial effectors as they possess predicted nuclease (Va16922 and Va18287) or pore-forming colicin-like (Va03175) domains (according to HHPred analysis [29]) that can mediate antibacterial toxicity. Moreover, the genes encoding for these three proteins were immediately upstream of small open reading frames (ORFs) that could encode for their cognate immunity proteins (Va16927, Va18282, and Va03180, respectively). These three putative effector/immunity pairs were encoded outside of the VaT6SS2 gene cluster (Fig 2A). To test whether Va16922/7, Va18287/2, and Va03175/80 are VaT6SS2 effector/immunity pairs, we monitored the ability of a V. alginolyticus wild-type strain, which is immune against self-intoxication, to kill strains with deletions in the putative effector/immunity gene pairs. Indeed, the wild-type strain was able to kill strains with deletions in va16922-7 and va18287-2 when co-cultured under VaT6SS2 inducing conditions (Fig 2B and 2C), indicating that deletion of these bicistronic units resulted in loss of immunity against self-intoxication. However, a strain deleted for va03175-80 was still immune against self-intoxication (Fig 2D), suggesting that either Va03175/80 are not an effector/immunity pair, or that there is an additional immunity gene. Using Va03180 as template, we performed a BLAST search to look for possible redundant immunity proteins in V. alginolyticus 12G01. We identified Va03170, encoded by the gene immediately upstream of the putative effector Va03175, as a homolog of Va03180 (65% identity). Therefore, we generated a strain deleted for the putative effector and the two homologous putative immunity genes, Δva03170-80. As predicted, the wild-type strain was able to kill the Δva03170-80 strain when co-cultured under VaT6SS2 inducing conditions (Fig 2D), indicating that va03170 and va03180 encode for redundant immunity proteins against Va03175-medited toxicity. In all cases, inactivation of VaT6SS2 by deletion of hcp2, or deletion of the effector/immunity pair in the attacking strain, resulted in loss of self-intoxication indicating that the toxic effectors were delivered by VaT6SS2 and encoded within these bicistronic units. Moreover, exogenous expression of the putative immunity proteins from a plasmid in the prey strains deleted for the effector/immunity pairs restored immunity against self-intoxication (Fig 2). Importantly, expression of either Va03170 or Va03180 from a plasmid restored immunity against self-intoxication in the Δva03170-80 strain indicating they are indeed redundant immunity proteins against Va03175-mediated toxicity (Fig 2D and S2 Fig). Taken together, these results demonstrate that Va16922/7, Va18287/2, and Va03175/80/70, are effector/immunity pairs of VaT6SS2.

Bottom Line: Using comparative proteomics and genetics, we identified their effector repertoires.We also showed that the T6SS2 secretes at least three antibacterial effectors.We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are "orphan" effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness.

No MeSH data available.