A phospholipase A<sub>1</sub> antibacterial Type VI secretion effector interacts directly with the C‐terminal domain of the VgrG spike protein for delivery

Contractile phage tails are powerful cell puncturing nanomachines that have been co-opted by bacteria for self-defense against both bacteria and eukaryotic cells. The tail of phage T4 has long served as the paradigm for understanding contractile tail-like systems despite its greater complexity compared with other contractile-tailed phages. Here, we present a detailed investigation of the assembly of a "simple" contractile-tailed phage baseplate, that of Escherichia coli phage Mu. By coexpressing various combinations of putative Mu baseplate proteins, we defined the required components of this baseplate and delineated its assembly pathway. We show that the Mu baseplate is constructed through the independent assembly of wedges that are organized around a central hub complex. The Mu wedges are comprised of only three protein subunits rather than the seven found in the equivalent structure in T4. Through extensive bioinformatic analyses, we found that homologs of the essential components of the Mu baseplate can be identified in the majority of contractile-tailed phages and prophages. No T4-like prophages were identified. The conserved simple baseplate components were also found in contractile tailderived bacterial apparatuses, such as type VI secretion systems, Photorhabdus virulence cassettes, and R-type tailocins. Our work highlights the evolutionary connections and similarities in the biochemical behavior of phage Mu wedge components and the TssF and TssG proteins of the type VI secretion system. In addition, we demonstrate the importance of the Mu baseplate as a model system for understanding bacterial phage tail-derived systems.baseplate | assembly | contractile tail | phage Mu

Section: Discussionsupporting

confidence: 70%

Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems

Buttner

Maxwell

et al. 2016

“…However, whether this linker sequence is required for Tap-1 binding to load effector on VgrG1 spike has remained unknown. Interestingly, Flaugnatti et al recently reported the direct binding of Tle1 phospholipase effector with C terminus of VgrG in E. coli Sci-1 T6SS without bridging by an adaptor protein (11). Taken together, these recent reports and our new findings indicate that the divergence in C-terminus sequences have allowed distinct mechanisms for effector loading and delivery among VgrG proteins.…”

Section: Discussionsupporting

confidence: 61%

“…Recent studies further identified a DUF4123-domaincontaining protein, Tap-1/Tec, that is required for loading a specific effector onto cognate VgrG for delivery in V. cholerae (32,33). Interestingly, the C terminus of VgrG in Escherichia coli Sci-1 T6SS interacts directly with the Tle1 phospholipase effector for delivery without bridging by an adaptor protein (11). However, the detailed molecular determinants and underlying mechanisms of VgrG-conferred effector transport specificity have not been elucidated.…”

Section: Significancementioning

confidence: 99%

“…A number of T6SS-secreted effectors were recently found to have contact-dependent antibacterial activity in various bacteria (1). The biochemical functions of characterized effectors include the cell wall-degrading enzymes (2-5), nuclease (6)(7)(8), and membrane lipid-degrading phospholipase (9)(10)(11). These diverse toxin effectors are widespread superfamilies in the bacterial kingdom and therefore likely have a general antibacterial strategy against competitor bacterial cells.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

147

273

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

“…T6SS effectors are delivered into target cells by a cargo mechanism using the Hcp hexamer or the VgrG/PAAR spike as a carrier (10,23,(42)(43)(44)(45). The SPI-6 T6SS encodes two distinct Hcp proteins, STM0276 and STM0279, hereafter called Hcp1 and Hcp2, respectively.…”

Section: S Typhimurium T6ss Kills Microbiota Members and Is Enhanced Bymentioning

confidence: 99%

Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut

Sana

Flaugnatti

Lugo

et al. 2016

Self Cite

298

292

The mammalian gastrointestinal tract is colonized by a high-density polymicrobial community where bacteria compete for niches and resources. One key competition strategy includes cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), a multiprotein needle-like apparatus that injects effector proteins into prokaryotic and/or eukaryotic target cells. However, the contribution of T6SS antibacterial activity during pathogen invasion of the gut has not been demonstrated. We report that successful establishment in the gut by the enteropathogenic bacterium Salmonella enterica serovar Typhimurium requires a T6SS encoded within Salmonella pathogenicity island-6 (SPI-6). In an in vitro setting, we demonstrate that bile salts increase SPI-6 antibacterial activity and that S. Typhimurium kills commensal bacteria in a T6SS-dependent manner. Furthermore, we provide evidence that one of the two T6SS nanotube subunits, Hcp1, is required for killing Klebsiella oxytoca in vitro and that this activity is mediated by the specific interaction of Hcp1 with the antibacterial amidase Tae4. Finally, we show that K. oxytoca is killed in the host gut in an Hcp1-dependent manner and that the T6SS antibacterial activity is essential for Salmonella to establish infection within the host gut. Our findings provide an example of pathogen T6SS-dependent killing of commensal bacteria as a mechanism to successfully colonize the host gut.