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1.C.107 The Import Subunit A of the Insecticidal Toxin Complex (ITC-A) Family

Toxin complex (Tc) proteins comprise a class of molecules, first identified in the nematode-associated bacterium Photorhabdus luminescens. Toxin complex (Tc) proteins characteristically belong to one of three distinct classes with at least one subunit from each of the three classes combining to form a complex with potent insecticidal activity. The Tc classes have been designated A, B, and C, and the P. luminescens Tc proteins can thus be classified as either TcA-like (TcaA1, TcaB1, TcbA, TccA and TcdA1), TcB-like (TcaC1 and TcdB1), or TcC-like (TccC1 and TccC2) (Bedford and Clarke 2009). Genes encoding Tc-like proteins have subsequently been identified in many insect-active bacteria including Serratia entomophila (tcA, sepA; tcB, sepB; tcC, sepC) (Wysocka et al. 2006) and the nematode-associated bacterium Xenorhabdus nematophila (tcA, xptA1, xptA2; tcB, xptC1; tcC, xptB1) (Deng et al. 2010), while the completion of bacterial genome sequencing projects such as Yersinia pestis C092 (Sanchez et al. 2010) and Pseudomonas syringae pv. tomato DC3000 (Hong et al. 2010) has revealed the presence of additional putative insecticidal tc genes. TcA is a determinant of target host range.

TcA of an insecticidal X. nematophila Tc binds to solubilized insect midgut brush border membranes (Nishida et al. 2009). TcC-like proteins are thought to represent the main toxin components of the Tc family (Wysocka et al., 2006). Two P. luminescens TcC-like toxins, TccC3 and TccC5, trigger ADP ribosylation and RhoA GTPase activation.

A model for Tc-induced toxicity has been proposed in which the TcA component selectively makes contact with the target cell wall, following which the TcC components are internalised to the cytosol (Gonsalvez et al. 2006). The structure of the oligomeric TcA-like X. nematophila protein XptA1 was solved in the absence of its TcB and TcC partners at moderate resolution (Nishida et al., 2009). The structure revealed a tetrameric cage-like assembly, proposed to encapsulate the TcB and TcC components (Landsberg et al. 2011).

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. Translocation of TcC into the cell is mediated by the TcA and TcB components. Gatsogiannis et al. (2013) showed that TcA in (TcdA1) forms a transmembrane pore. Its structures in the prepore and pore states were determined by cryoelectron microscopy. They found that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter, surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 revealed large conformational changes during membrane insertion suggesting a novel syringe-like mechanism of protein translocation. These results demonstrated how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. The proposed mechanism may be characteristic of the whole ABC-type toxin family. 

Using electron cryomicroscopy, Gatsogiannis et al. 2016 determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. The new structure, compared with the previous structure of TcdA1 in the prepore state, shows that the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.

Homologues of the B and C subunits are found in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggesting that these toxins are important in a range of different hosts, including man (Yang and Waterfield 2013). TccC3 ADP-ribosylates actin at Thr148 promoting polymerization and aggregation of intracellular F-actin, leading to inhibition of several cellular functions, such as phagocytosis (Lang et al. 2016).

The high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex have been solved (Meusch et al. 2014). The structures reveal that in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed β-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation (Meusch et al. 2014).

The generalized reaction catalyzed by TcTCs is:

Toxin protein and small molecules (out) → Toxin protein and small molecules (in)

References associated with 1.C.107 family:

Bedford, M.T. and S.G. Clarke. (2009). Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33: 1-13. 19150423
Deng, X., L. Gu, C. Liu, T. Lu, F. Lu, Z. Lu, P. Cui, Y. Pei, B. Wang, S. Hu, and X. Cao. (2010). Arginine methylation mediated by the Arabidopsis homolog of PRMT5 is essential for proper pre-mRNA splicing. Proc. Natl. Acad. Sci. USA 107: 19114-19119. 20956294
Gatsogiannis, C., A.E. Lang, D. Meusch, V. Pfaumann, O. Hofnagel, R. Benz, K. Aktories, and S. Raunser. (2013). A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature 495: 520-523. 23515159
Gatsogiannis, C., F. Merino, D. Prumbaum, D. Roderer, F. Leidreiter, D. Meusch, and S. Raunser. (2016). Membrane insertion of a Tc toxin in near-atomic detail. Nat Struct Mol Biol. [Epub: Ahead of Print] 27571177
Gonsalvez, G.B., T.K. Rajendra, L. Tian, and A.G. Matera. (2006). The Sm-protein methyltransferase, dart5, is essential for germ-cell specification and maintenance. Curr. Biol. 16: 1077-1089. 16753561
Hong, S., H.R. Song, K. Lutz, R.A. Kerstetter, T.P. Michael, and C.R. McClung. (2010). Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 107: 21211-21216. 21097700
Hurst, M.R., T.R. Glare, T.A. Jackson, and C.W. Ronson. (2000). Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens. J. Bacteriol. 182: 5127-5138. 10960097
Landsberg, M.J., S.A. Jones, R. Rothnagel, J.N. Busby, S.D. Marshall, R.M. Simpson, J.S. Lott, B. Hankamer, and M.R. Hurst. (2011). 3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity. Proc. Natl. Acad. Sci. USA 108: 20544-20549. 22158901
Nishida, K.M., T.N. Okada, T. Kawamura, T. Mituyama, Y. Kawamura, S. Inagaki, H. Huang, D. Chen, T. Kodama, H. Siomi, and M.C. Siomi. (2009). Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines. EMBO. J. 28: 3820-3831. 19959991
Sanchez, S.E., E. Petrillo, E.J. Beckwith, X. Zhang, M.L. Rugnone, C.E. Hernando, J.C. Cuevas, M.A. Godoy Herz, A. Depetris-Chauvin, C.G. Simpson, J.W. Brown, P.D. Cerdán, J.O. Borevitz, P. Mas, M.F. Ceriani, A.R. Kornblihtt, and M.J. Yanovsky. (2010). A methyl transferase links the circadian clock to the regulation of alternative splicing. Nature 468: 112-116. 20962777
Wysocka, J., C.D. Allis, and S. Coonrod. (2006). Histone arginine methylation and its dynamic regulation. Front Biosci 11: 344-355. 16146736