3.A.23 The Type VI Symbiosis/Virulence Secretory System (T6SS) Family
Some Vibrio cholerae strains do not cause cholera and instead cause human infections. One such strain possesses on its small chromosome a 15 cistron operon that catalyze protein secretion A hydrophobic N-terminal sequence is not required (Pukatzki et al., 2006; Filloux et al., 2008). The protein substrates can be secreted into the extracellular medium, into eukaryotic cells, and into other bacteria. The genes have been named vasA-L (virulence-associated secretion) or the type VI secretion system (T6SS). At least some of the vas genes are required for cytotoxicity by a contact-dependent mechanism.
The Type VI secretion system (T6SS) is an injection apparatus that uses a spring-like mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is assured by a dedicated trans-envelope channel complex. This membrane complex (MC) consists of the TssJ lipoprotein, and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative E. coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Santin et al. 2019 showed that the PGB domain is the only functional domain of TagL, and that the N-terminal TMS mediates contact with the TssL transmembrane helix. TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking.
Many Gram-negative bacteria have homologous genes as well as potential effector genes. The V. cholerae Vas system may secrete proteins called Hep, VrgG1, VrgG2 and VrgG3. VasK (1181 aas) resembles IcmF (973 aas; AAU26555) of the Legionella pneumophila Dot system throughout most of its length. Similarly, VasF (257 aas) resembles IcmH (261 aas; AAU26556) of the Dot system. Throughout most of its length and like the VasF protein, IcmH shows 4 peaks of hydrophobicity. VasF and IcmH also show sequence similarity with portions of the OmpA/MotB proteins, possibly possessing a peptidoglycan binding domain. While all 15 cistrons of the vas operon are presented under TC# 3.A.23.1.1, it is unlikely that they all function directly in transport. For example, VasG resembles the E. coli ClpB ATP-dependent, stress-induced, heat shock chaperone protein that uses ATP hydrolysis to unfold denatured proteins in conjunction with DnaJK and GrpE. Moreover, VasH resembles a sigma-54-dependent transcriptional activator, and a sigma-54 is encoded downstream of the vas operon. Only VasA, VasF and VasK have been shown to promote virulence. ATP hydroloysis is involved in the energization process (Pukatzki et al. 2006; Basler and Mekalanos 2012).
In Vibrio cholerae, the T6SS secretes three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. Protein structure search algorithms predict that VgrG-related proteins assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate 'tail spike' of Escherichia coli bacteriophage T4. VgrG-1 interacts with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form (Pukatzki et al. 2007). Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, the VgrG components of the T6SS apparatus may assemble a 'cell-puncturing device' analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells. As noted above, the VgrGs, share structural features with the cell-puncturing device of the T4 bacteriophage. Possibly, they are used in a similar fashion by bacteria to puncture host cell membranes and insert the T6SS apparatus into the host cytosol. A number of VgrGs contain C-terminal extensions with effector-domains. Thus, the VISP may translocate soluble effectors as well as VgrG effector-domains (Pukatzki et al. 2009). The type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin (Leiman et al. 2009).
Several Gram-negative bacterial pathogens and symbions have T6SSs (Saier 2006). In Edwardsiella tarda, an E. tarda virulent protein (EVP) gene cluster is involved in protein secretion, and the entire EVP cluster codes for a T6SS. All the 16 EVP genes were mutagenized; the secretion of EvpP was dependent on 13 EVP proteins including EvpC (a homologue of Hcp) and EvpI but not EvpD and EvpJ. The 16 EVP proteins were grouped according to their functions and cellular locations. The first group comprises 11 non-secreted and possibly intracellular apparatus proteins. Among them, EvpO, a putative ATPase which contains a Walker A motif, shows possible interactions with three EVP proteins (EvpA, EvpL and EvpN). The second group includes three secreted proteins (EvpC, EvpI and EvpP). The secretion of EvpC and EvpI is mutually dependent, and they are required for the secretion of EvpP. An interaction between EvpC and EvpP was demonstrated. Two proteins (EvpD and EvpJ) are not required for the T6SS-dependent secretion (Zheng and Leung 2007).
Protein secretion by the T6SS of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheaths. Sheaths of the T6SS cycle between assembly, quick contraction, disassembly and re-assembly (Basler et al. 2012). Whole-cell electron cryotomography showed that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. Possibly, contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.
Type 6 secretion dynamics within and between bacterial cells have been measured (Basler and Mekalanos 2012). ClpV imaging provided evidence that P. aeruginosa likely recycles T6SS membrane base plate components and can sense T6SS activity in nearby cells (Basler and Mekalanos 2012). Because T6SS dueling events were spatially and temporally linked, they likely mark the exact location of T6SS translocation of protein components (e.g., VgrG and/or effector proteins) between cells. T6SS dueling may reflect social interactions between heterologous T6SS+ species that coexist in the same niche. One of the substrates of a T6SS in Dickeya dadantii is an Rhs protein that mediates intercellular competition with other bacteria (Koskiniemi et al. 2013). This protein carries polymorphic C-terminal toxin domains which are deployed to inhibit the growth of neighboring cells. They also contain immunity protein domains that neutralize the toxins to protect the producing cells. In Bacillus subtilis,a distantly related WapA proteini is exported via the general secretory pathway and delivers the tRNase toxins into neighboring target cells (Koskiniemi et al. 2013).
The T6SS is a dynamic organelle that bacteria use to target prey cells for inhibition via translocation of effector proteins. Opposed T6SS organelles in adjacent sister cells of Pseudomonas aeruginosa give rise to 'T6SS dueling' and likely reflect a biological process that is driven by T6SS antibacterial attack. Basler et al. (2013) have shown T6SS dueling behavior influences the ability of P. aeruginosa to prey upon heterologous bacterial species. T6SS-dependent killing of either Vibrio cholerae or Acinetobacter baylyi is greatly stimulated by T6SS activity occurring in those prey species. Thus, in P. aeruginosa, T6SS organelle assembly and lethal counterattack are regulated by a signal that corresponds to the point of attack of the T6SS apparatus elaborated by a second aggressive T6SS+ bacterial cell.
Bacterial T6SSs are akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The P. aeruginosa H1-T6SS is involved in bacterial killing and the delivery of three toxins, Tse1-3. Hachani et al. 2014 demonstrated the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C-terminus carries a toxic activity, from which the producing bacterium is protected by immunity. VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which has the characteristics of superfamily nuclease 2 toxins. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes; they encode putative toxins. These associations support the notion of a form of secretion system in which VgrG acts as the carrier (Hachani et al. 2014).
Natural competence for transformation allows horizontal gene transfer and occurs through the uptake of external DNA followed by integration into the genome (see TC# 3.A.11). Borgeaud et al. 2015 showed that the T6SS, which serves as a predatory killing device, is part of the competence regulon in Vibrio cholerae (see TC# 3.A.11.2.3). The T6SS-encoding gene cluster is under positive control by the competence regulators, TfoX and QstR, and is induced by growth on chitinous surfaces (Borgeaud et al. 2015). Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae.
The generalized nomenclature for T6SSs uses the Tss nomenclature, TssA - M, for the core constituents of a system (Cianfanelli et al. 2016). TssJLM form a membrane complex, solved at 11.6 Å, showing a structure with 5-fold symmetry which extends from a substantial base in the cytoplasm and inner membrane, through periplasmic arches, into a periplasmic tip complex contacting the outer membrane. This complex is assembled sequentially from TssJ, TssM, and TssL. Large conformational changes on assembly of an active T6SS cause the C-terminus of TssM to cross the outer membrane, forming a transient pore through which the puncturing spike passes (Cianfanelli et al. 2016).
Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Böck et al. 2017 used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are widespread. (Böck et al. 2017). T6SSs and their effectors have been reviewed (Russell et al. 2014; Silverman et al. 2012; Galán and Waksman 2018; Clemens et al. 2018; Lennings et al. 2018; Iacob et al. 2018).
prePAAR is a conserved motif found in over 6000 putative TMS-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMSs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMS-chaperone complexes from Salmonella enterica Typhimurium and Pseudomonas aeruginosa, respectively, revealed that Eag chaperones mimic transmembrane helical packing to stabilize effector TMSs (Ahmad et al. 2020). In addition to participating in the chaperone-TMD interface, prePAAR residues mediate effector-VgrG spike interactions. Thus, mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors have been identified.
The reaction catalyzed by most T6SSs is:
Protein and/or DNA (cytoplasm of the donor) → Protein and/or DNA (cytoplasm of the recipient).