Lytic Activity of the Vibrio cholerae Type VI Secretion Toxin VgrG-3 Is Inhibited by the Antitoxin TsaB

Brooks, Teresa M.; Unterweger, Daniel; Bachmann, Verena; Kostiuk, Benjamin; Pukatzki, Stefan

doi:10.1074/jbc.m112.436725

Cited by 157 publications

(202 citation statements)

References 26 publications

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“…In some cases, the same effector can function in bacterial antagonism and also alter cell-signalling pathways in eukaryotic cells (Jiang et al, 2014). Also, 'evolved' VgrGs have been described that contain various C-terminal extensions leading, for instance, to actin cross-linking or actin-ADP ribosylation in eukaryotic cells (Brooks et al, 2013;Pukatzki et al, 2007;Suarez et al, 2010), and host cell fusion presumably to facilitate intercellular bacterial spreading (Schwarz et al, 2014;Toesca et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

Aubert

Valvano

2015

Microbiology

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The Gram-negative bacterial type VI secretion system (T6SS) delivers toxins to kill or inhibit the growth of susceptible bacteria, while other secretion systems target eukaryotic cells. Deletion of atsR, a negative regulator of virulence factors in B. cenocepacia K56-2, increases T6SS activity. Macrophages infected with a K56-2 DatsR mutant display dramatic alterations in their actin cytoskeleton architecture that rely on the T6SS, which is responsible for the inactivation of multiple Rho-family GTPases by an unknown mechanism. We employed a strategy to standardize the bacterial infection of macrophages and densitometrically quantify the T6SS-associated cellular phenotype, which allowed us to characterize the phenotype of systematic deletions of each gene within the T6SS cluster and ten vgrG genes in K56-2 DatsR. None of the genes from the T6SS core cluster nor the individual vgrG genes were directly responsible for the cytoskeletal changes in infected cells. However, a mutant strain with all vgrG genes deleted was unable to cause macrophage alterations. Despite not being able to identify a specific effector protein responsible for the cytoskeletal defects in macrophages, our strategy resulted in the identification of the critical core components and accessory proteins of the T6SS assembly machinery and provides a screening method to detect T6SS effectors targeting the actin cytoskeleton in macrophages by random mutagenesis.

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Section: Introductionmentioning

confidence: 99%

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

Aubert

Valvano

2015

Microbiology

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“…It may feature the effector function at the C-terminal domain. Such "evolved VgrGs" include Vibrio cholerae VgrG-1, with an actin cross-linking domain; Aeromonas hydrophila VgrG1, with an actin-ADP ribosylating VIP-2 domain; and V. cholerae VgrG-3, with glycoside hydrolase activity to target peptidoglycan (9,(25)(26)(27). Furthermore, VgrG may function as a carrier for effector delivery by binding directly with specific effectors carrying the proline-alanine-alanine-arginine (PAAR) domain (28,29).…”

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confidence: 99%

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

Bondage

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

144

273

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Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity.type VI secretion system | VgrG | DNase effector | interbacterial competition | Agrobacterium tumefaciens

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“…The product was cloned between the NcoI and XhoI restriction sites of the expression vector pET-28a (Novagen) with a hexahistidine tag at the C-terminus as described previously (Brooks et al, 2013). After sequencing, the plasmid was transformed into Escherichia coli BL21 (DE3) competent cells (Novagen, USA).…”

Section: Cloning and Expressionmentioning

confidence: 99%

“…Subsequently, it was reported that TsiV3 (TsaB) directly binds to and inhibits effector protein VgrG-3 in a type II toxin-antitoxin (TA) manner and thus presents immunity to the toxic activity of VgrG-3 (Brooks et al, 2013). As a novel T6SS immunity protein, TsiV3 does not share high sequence identity with other immunity proteins of known structure (Q9I2Q0, C6BHF3, Q8ZRL5, Q9HYC4, Q4KC91 and Q9I0D9; Shang et al, 2012;Dong, Zhang et al, 2013;Zhang et al, 2013;Wang et al, 2013;Whitney et al, 2013;Li et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…VgrG proteins possess additional Cterminal domains that act as effectors to attack prey cells, as found in VgrG-1 and VgrG-3 of the three VgrG proteins (VgrG-1, VgrG-2 and VgrG-3) encoded by the T6SS genes of V. cholerae (Pukatzki et al, 2007). Previous studies have shown that VgrG-3 from V. cholerae contains a peptidoglycan-binding domain (PG1) at the C-terminus and that this domain plays an important role in hydrolyzing the cell wall of Gram-negative bacteria, thereby granting V. cholerae a competitive advantage against adjacent cells (Brooks et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Crystallization and preliminary X-ray study of TsiV3 fromVibrio cholerae

Zhang

Liu

et al. 2014

Acta Cryst Sect F

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The bacterial type VI secretion system (T6SS), a dynamic organelle, participates in microbial competition by transporting toxic effector molecules to neighbouring cells to kill competitors. TsiV3, a recently defined T6SS immunity protein in Vibrio cholerae, possesses self-protection against killing by T6SS predatory cells by directly binding to and inhibiting their effector protein VgrG-3. Structural information about TsiV3 could help to illuminate its specific mechanism. In this study, TsiV3 from V. cholerae was cloned, expressed and crystallized and single-crystal X-ray diffraction data sets were collected to a resolution of 2.55 Å . Specifically, the crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 73.3, b = 78.12, c = 106.18 Å . Matthews coefficient calculations indicated that the crystal may contain six TsiV3 molecules in one asymmetric unit, with a V M value of 2.25 Å 3 Da À1 and a solvent content of 45.42%.

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Lytic Activity of the Vibrio cholerae Type VI Secretion Toxin VgrG-3 Is Inhibited by the Antitoxin TsaB

Cited by 157 publications

References 26 publications

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

Crystallization and preliminary X-ray study of TsiV3 fromVibrio cholerae

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