Structure and function of the type VI secretion system in bacterial pathogens
This joint PhD project is based at the Shanghai Jiao Tong University with a minimum 12 month stay at The University of Melbourne.
The type VI secretion system (T6SS) is a microbial weapon employed by many pathogens to directly inject toxins into recipient prokaryotic and eukaryotic cells. T6SS structure consists of a long (~1 um) contractile sheath-tube that is anchored to a transmembrane-baseplate complex. Molecular details of T6SS assembly remain elusive. In this joint project, we plan to combine the exceptional strength of structural biology at UofM and T6SS biology at SJTU to tackle the assembly mechanism, using structural and genetics tools such as cryo-ET and mutagenesis to examine the assembly details and determine key gene functions in model pathogens.
Microbial pathogenesis poses a serious threat to public health. Of the diverse virulence mechanisms found in gram-negative pathogens, the type VI secretion system (T6SS) is unique for its dual role in killing bacterial competitors and eukaryotic cells by injecting toxins directly from a donor cell to a recipient cell. Therefore, the T6SS plays a key role in microbiome composition and functions.
Evolved from contractile bacteriophage tails, the T6SS is featured with a long (~1 um) double tubular sheath- tube structure that is connected to a transmembrane complex and a baseplate structure. Assembly of the sheath-tube initiates from the baseplate and extends to the other side of the cell, involving at least 13 conserved structural proteins. How these multi-protein complexes are coordinated spatially and temporally remains elusive.
The T6SS is unequivocally shown as a contractile secretion system through the collaborative work by the Grant Jensen group at Caltech and the John Mekalanos group at Harvard University. The two applicants of this proposal were former postdoctoral trainees of those two prestigious laboratories; the UOM applicant for Debnath Ghosal from the Jensen lab and the SJTU applicant for Tao Dong from the Mekalanos lab. The mutual interest of the two applicants to push for deeper understanding of secretion systems build a solid foundation for the proposal. In this joint project, we plan to combine the strength of structural biology at UoM and bacterial genetics at SJTU to tackle the assembly process using several important model pathogens and a panel of genetic mutants.
This joint proposal will address three key questions of the T6SS assembly:
- How are eﬀectors loaded in situ and involved in T6SS assembly?
- What are the exact functions of the T6SS membrane-baseplate complex (MBC) components?
- Are eﬀector-cognate chaperones localized together with eﬀectors within the MBC?
Our hypothesis is that T6SS assembly initiation is controlled by the sequential interaction of eﬀectors with speciﬁc components of the membrane-baseplate complex.
To address these questions, we will employ a plethora of structural, biochemical and genetics tools and a panel of T6SS eﬀector mutants of V. cholerae and P. aeruginosa, two T6SS model species. We have three speciﬁc aims:
- Detect and determine eﬀector state (folded or unfolded) within the precontraction membrane-baseplate complex. To date, this remains elusive.
- Determine the function of diﬀerent membrane- baseplate complex (MBC) components on T6SS assembly. Our preliminary results show that some MBC mutants show sheath-tube aggregation while the others could assemble infrequent sheath-tube structures. What are the structures of the aggregates and mutant sheath-tubes?
- Direct visualization of the T6SS eﬀector-chaperone pairs during the assembly and follow the secretion process.
At the UoM, the Ghosal group has strong expertise in using electron cryotomography to characterize bacterial secretion systems including the type II secretion (Ghosal et al., 2019, Nature Microbiology) and type IV secretion (Ghosal et al., 2019, Nature Microbiology). At the SJTU, the Dong lab has established an international track record on the T6SS eﬀector function and secretion mechanisms, including the discoveries of eﬀector chaperones (Liang et al., 2015, PNAS), PAAR-dependent secretion and eﬀector (Burkinshaw et al., 2018, Nature Microbiology), and self-cleaved eﬀectors with intramolecular chaperones (Pei et al., 2020, Nature Communications). Accordingly, we propose to clearly separate the tasks between our two groups. The Ghosal group will take the lead in cryotomography imaging of the T6SS structures while the Dong group will primarily focus on research eﬀorts of mutant construction, biochemical assays and ﬂuorescence microscopy.
- Burkinshaw, B.J., Liang, X., Wong, M., Le, A.N.H., Lam, L., and Dong, T.G. (2018). A type VI secretion system eﬀector delivery mechanism dependent on PAAR and a chaperone–co-chaperone complex. Nat. Microbiol. 3, 632– 640.
- Ghosal, D., Kim, K.W., Zheng, H., Kaplan, M., Truchan, H.K., Lopez, A.E., McIntire, I.E., Vogel, J.P., Cianciotto, N.P., and Jensen, G.J. (2019a). In vivo structure of the Legionella type II secretion system by electron cryotomography. Nat. Microbiol. 4, 2101–2108.
- Ghosal, D., Jeong, K.C., Chang, Y.-W., Gyore, J., Teng, L., Gardner, A., Vogel, J.P., and Jensen, G.J. (2019b). Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS. Nat. Microbiol. 4, 1173–1182.
- Liang, X., Moore, R., Wilton, M., Wong, M.J.Q., Lam, L., and Dong, T.G. (2015). Identiﬁcation of divergent type VI secretion eﬀectors using a conserved chaperone domain. Proc. Natl. Acad. Sci. 112, 9106–9111.
- Liang, X., Kamal, F., Pei, T.-T., Xu, P., Mekalanos, J., and Dong, T.G. (2019). An onboard checking mechanism ensures eﬀector delivery of the type VI secretion system in Vibrio cholerae. Proc. Natl. Acad. Sci. 116, 23292–23298.
- Pei, T.-T., …, Dong, T.G. (2020). Intramolecular chaperone-mediated secretion of an Rhs eﬀector toxin by a type VI secretion system. Nat. Commun. 11, 1865.
The project will be complemented by the project onInvestigating the molecular mechanism of effector secretion by the bacterial type IV secretion system and the collaboration will ensure a successful completion of the project.
Professor Tao Dong (Shanghai Jiao Tong University)
Dr Debnath Ghosal (The University of Melbourne)