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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.


Related in: MedlinePlus

Va16152/Va16147, Va01565/Va01560, and Va01435/Va01430 are VaT6SS1 effector/immunity pairs.(A) Schematic representation of the VaT6SS1 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) Va16152/Va16147, (C) Va01565/Va01560, and (D) Va01435/Va01430. (E) Viability counts of prey strains before (0h) and after (4h) co-culture with V. alginolyticus 12G01 Δhcp2/Δhns (T6SS1+) or Δhcp1/Δhcp2/Δhns (T6SS1-) strains. Dashed line marks the assay detection limit. 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.g003: Va16152/Va16147, Va01565/Va01560, and Va01435/Va01430 are VaT6SS1 effector/immunity pairs.(A) Schematic representation of the VaT6SS1 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) Va16152/Va16147, (C) Va01565/Va01560, and (D) Va01435/Va01430. (E) Viability counts of prey strains before (0h) and after (4h) co-culture with V. alginolyticus 12G01 Δhcp2/Δhns (T6SS1+) or Δhcp1/Δhcp2/Δhns (T6SS1-) strains. Dashed line marks the assay detection limit. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).

Mentions: As predicted by the presence of the MIX motif, two of the identified secreted proteins, Va16152 and Va01565, were previously classified by us as putative T6SS MIX-effectors [16]. Va16152 contains an N-terminal MIX motif belonging to the MIX IV clan and a C-terminal pore-forming colicin-like domain (according to HHPred analysis [29]), and is encoded outside of the VaT6SS1 gene cluster. Va01565, which is encoded at the beginning of the VaT6SS1 gene cluster, contains a MIX motif belonging to the MIX I clan and is a homolog of the V. parahaemolyticus MIX-effector VP1388 [16]. Another VaT6SS1 secreted protein, Va01435, is encoded at the end of the VaT6SS1 gene cluster and is predicted to contain an N-terminal LysM peptidoglycan-binding domain followed by a peptidoglycan (PG) hydrolase domain and a lysozyme-like domain (according to HHPred analysis [29]). Moreover, the genes encoding for these three proteins were immediately upstream of small ORFs that could encode for their cognate immunity proteins (Va16147, Va01560, and Va01430, respectively) (Fig 3A). To test whether Va16152/47, Va01565/0, and Va01435/0 are VaT6SS1 effector/immunity pairs, we monitored the ability of a V. alginolyticus wild-type strain to kill strains with deletions in the putative effector/immunity gene pairs. As shown in Fig 3B–3D, the wild-type strain was able to inhibit the growth of strains with deletions in va16152-47, va01565-0 and va01435-0 when co-cultured under VaT6SS1 inducing conditions, and inactivation of VaT6SS1 by deletion of hcp1 or deletion of the effector/immunity pairs in the attacking strains resulted in increased growth of the prey strains. Moreover, exogenous expression of the putative immunity proteins Va16147 and Va01560 from a plasmid, but not of Va01430, also resulted in increased growth of the prey strains deleted for the cognate effector/immunity pairs. It is possible that the inability of the plasmid encoding for Va01430 to complement the deletion resulted from poor expression of Va01430 under the tested conditions.


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)

Va16152/Va16147, Va01565/Va01560, and Va01435/Va01430 are VaT6SS1 effector/immunity pairs.(A) Schematic representation of the VaT6SS1 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) Va16152/Va16147, (C) Va01565/Va01560, and (D) Va01435/Va01430. (E) Viability counts of prey strains before (0h) and after (4h) co-culture with V. alginolyticus 12G01 Δhcp2/Δhns (T6SS1+) or Δhcp1/Δhcp2/Δhns (T6SS1-) strains. Dashed line marks the assay detection limit. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4549250&req=5

ppat.1005128.g003: Va16152/Va16147, Va01565/Va01560, and Va01435/Va01430 are VaT6SS1 effector/immunity pairs.(A) Schematic representation of the VaT6SS1 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) Va16152/Va16147, (C) Va01565/Va01560, and (D) Va01435/Va01430. (E) Viability counts of prey strains before (0h) and after (4h) co-culture with V. alginolyticus 12G01 Δhcp2/Δhns (T6SS1+) or Δhcp1/Δhcp2/Δhns (T6SS1-) strains. Dashed line marks the assay detection limit. Asterisks mark statistical significance between sample groups at t = 4h by an unpaired, two tailed student’s t-test (p<0.05).
Mentions: As predicted by the presence of the MIX motif, two of the identified secreted proteins, Va16152 and Va01565, were previously classified by us as putative T6SS MIX-effectors [16]. Va16152 contains an N-terminal MIX motif belonging to the MIX IV clan and a C-terminal pore-forming colicin-like domain (according to HHPred analysis [29]), and is encoded outside of the VaT6SS1 gene cluster. Va01565, which is encoded at the beginning of the VaT6SS1 gene cluster, contains a MIX motif belonging to the MIX I clan and is a homolog of the V. parahaemolyticus MIX-effector VP1388 [16]. Another VaT6SS1 secreted protein, Va01435, is encoded at the end of the VaT6SS1 gene cluster and is predicted to contain an N-terminal LysM peptidoglycan-binding domain followed by a peptidoglycan (PG) hydrolase domain and a lysozyme-like domain (according to HHPred analysis [29]). Moreover, the genes encoding for these three proteins were immediately upstream of small ORFs that could encode for their cognate immunity proteins (Va16147, Va01560, and Va01430, respectively) (Fig 3A). To test whether Va16152/47, Va01565/0, and Va01435/0 are VaT6SS1 effector/immunity pairs, we monitored the ability of a V. alginolyticus wild-type strain to kill strains with deletions in the putative effector/immunity gene pairs. As shown in Fig 3B–3D, the wild-type strain was able to inhibit the growth of strains with deletions in va16152-47, va01565-0 and va01435-0 when co-cultured under VaT6SS1 inducing conditions, and inactivation of VaT6SS1 by deletion of hcp1 or deletion of the effector/immunity pairs in the attacking strains resulted in increased growth of the prey strains. Moreover, exogenous expression of the putative immunity proteins Va16147 and Va01560 from a plasmid, but not of Va01430, also resulted in increased growth of the prey strains deleted for the cognate effector/immunity pairs. It is possible that the inability of the plasmid encoding for Va01430 to complement the deletion resulted from poor expression of Va01430 under the tested conditions.

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.


Related in: MedlinePlus