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3.A.23 The Type VI Symbiosis/Virulence Secretory Pathway (VISP) Family

Some Vibrio cholerae strains do not cause cholera and instead cause human infections by poorly understood mechanisms. One such strain possesses on its small chromosome a 15 cistron operon that encodes 15 proteins. They have been reported to catalyze protein secretion by a mechanism that does not require the presence of a hydrophobic N-terminal sequence (Pukatzki et al., 2006; Filloux et al., 2008). The protein substrates can be secreted into the extracellular medium, and possibly into eukaryotic cells. 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. 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, but homology could not be demonstrated. 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 with the first and last being most hydrophobic. VasF and IcmH also show some sequence similarity with portions of OmpA/MotB proteins, possibly possessing a peptidoglycan binding domain. While all 15 cistrons of the vas operon are presented below, 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 in E. coli. 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 VISP 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 type VI secretion systems (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. Lastly, two proteins (EvpD and EvpJ) are not required for the T6SS-dependent secretion (Zheng and Leung 2007).

Protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheaths. Time-lapse fluorescence light microscopy revealed that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly (Basler et al. 2012). Whole-cell electron cryotomography further 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 type VI secretion system 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 from toxicity.  In Bacillus subtilis, a distantly related WapA proteinis exported via the general secretory pathway and delivers the tRNase toxins into neighboring target cells (Koskiniemi et al. 2013).

The bacterial type VI secretion system (T6SS) is a dynamic organelle that bacteria use to target prey cells for inhibition via translocation of effector proteins. Time-lapse fluorescence microscopy has documented striking dynamics of opposed T6SS organelles in adjacent sister cells of Pseudomonas aeruginosa. Such cell-cell interactions have been termed 'T6SS dueling' and likely reflect a biological process that is driven by T6SS antibacterial attack. Basler et al. (2013) have shown T6SS dueling behavior strongly 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 type VI secretion systems (T6SS) are supra-molecular complexes akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas 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. Similarly, 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. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Thus, VgrGs are components specifically deliver effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes, and 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 is a common mode of 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 type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae (see 3.A.11.2.3). The T6SS-encoding gene cluster is under the positive control of 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 Type VI secretion systems uses the Tss nomenclature, TssA - M, for the core constituents of the 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 and a transient pore to be formed 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 more widespread than previously thought. These insights form the basis for understanding T6SS key concepts and exploring T6SS diversity (Böck et al. 2017).

The reaction catalyzed by most T6SS systems is:

Protein and/or DNA (cytoplasm of the donor) → Protein and/or DNA (cytoplasm of the recipient).

References associated with 3.A.23 family:

Basler, M. and J.J. Mekalanos. (2012). Type 6 secretion dynamics within and between bacterial cells. Science 337: 815. 22767897
Basler, M., B.T. Ho, and J.J. Mekalanos. (2013). Tit-for-Tat: Type VI Secretion System Counterattack during Bacterial Cell-Cell Interactions. Cell 152: 884-894. 23415234
Basler, M., M. Pilhofer, G.P. Henderson, G.J. Jensen, and J.J. Mekalanos. (2012). Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483: 182-186. 22367545
Böck, D., J.M. Medeiros, H.F. Tsao, T. Penz, G.L. Weiss, K. Aistleitner, M. Horn, and M. Pilhofer. (2017). In situ architecture, function, and evolution of a contractile injection system. Science 357: 713-717. 28818949
Borgeaud, S., L.C. Metzger, T. Scrignari, and M. Blokesch. (2015). Bacterial evolution. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science 347: 63-67. 25554784
Chang, Y.W., L.A. Rettberg, D.R. Ortega, and G.J. Jensen. (2017). In vivo structures of an intact type VI secretion system revealed by electron cryotomography. EMBO Rep 18: 1090-1099. 28487352
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Pukatzki, S., A.T. Ma, A.T. Revel, D. Sturtevant, and J.J. Mekalanos. (2007). Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc. Natl. Acad. Sci. USA 104: 15508-15513. 17873062
Pukatzki, S., A.T. Ma, D. Sturtevant, B. Krastins, D. Sarracino, W.C. Nelson, J.F. Heidelberg, and J.J. Mekalanos. (2006). Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. USA 103: 1528-1533. 16432199
Pukatzki, S., S.B. McAuley, and S.T. Miyata. (2009). The type VI secretion system: translocation of effectors and effector-domains. Curr. Opin. Microbiol. 12: 11-17. 19162533
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Shneider, M.M., S.A. Buth, B.T. Ho, M. Basler, J.J. Mekalanos, and P.G. Leiman. (2013). PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500: 350-353. 23925114
Zheng, J. and K.Y. Leung. (2007). Dissection of a type VI secretion system in Edwardsiella tarda. Mol. Microbiol. 66: 1192-1206. 17986187