Molecular mechanisms powering a bacterial toxin injection device. (360G-Wellcome-104634_Z_14_Z)

£750,000

Type III secretion systems (T3SSs) are essential devices in the virulence of many Gram-negative bacterial pathogens. They translocate bacterial virulence proteins into eukaryotic host cells to manipulate them during infection. T3SSs involved in virulence (vT3SSs) are related to bacterial flagella assembly apparatuses (fT3SSs). The importance of vT3SSs to human disease lead to their intensive study by many laboratories over the last two decades. My laboratory provided insights into the vT3SS bas e that spans both bacterial membranes and the periplasm, the external and hollow injection needle embedded in the base and its distal tip complex, which transforms itself into the translocation pore in the host cell membrane. We thus illuminated how vT3SSs are activated for protein translocation by physical contact of the needle tip with host cells, a key step in infection initiation. However, our understanding of the other central function of T3SSs, the translocation process remains incomplete . This is energised solely by the cytoplasmic and inner membrane export apparatus (CIMEA), which mediates the passage of substrates across the bacterial inner membrane (IM). The cytoplasmic export apparatus (CEA) comprises an ATPase complex attached by the cytoplasmic or C-ring to the T3SS base. The inner membrane export apparatus (IMEA) probably forms the export pore inside the T3SS base. Only the IMEA is essential for export, for which it uses energy derived from the proton motive force (pmf). Export is also strongly enhanced by the ATPase. To enter the narrow channel traversing the needle or flagellum, substrates must carry a targeting motif and become unfolded. Such substeps were reconstituted in vitro. Furthermore, a substrate secretion hierarchy was established. But, it is unclear how to integrate this information with that on CIMEA energetics, component structure, and architecture in situ. Therefore, the overall mechanism of T3SS-mediated protein export remains obscure. To inv estigate this, we will focus on the IMEA and how it interfaces with the CEA. For this, we will work mainly on the T3SS with the best-understood CEA, the Salmonella fT3SS. As the CIMEA is the most conserved part of T3SSs what we find there is directly relevant to vT3SSs. Our questions are: 1) How is the native IMEA organised structurally and in terms of component stoichiometries and interactions? 2) How does it use the pmf to energise protein export? How is transport enhanced by ATP hydrolysis and the CEA? 3) What is the path of substrates through the CIMEA? 4) How does the CIMEA change to allow secretion of different substrates at different rates? To answer them, I envision using novel genetic strategies to overexpress sufficient IMEA for biochemical and structural characterisation. We will then develop an in vitro assay for T3SS-mediated protein export to dissect CIMEA function. We will complement this with in vivo studies of CIMEA morphology, stoichiometry, dynamics using conve ntional and super-resolution light microscopy (SRLM) on the Shigella vT3SS, where we have appropriate tools and mutants.

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Grant Details

Amount Awarded 750000
Applicant Surname Blocker
Approval Committee Science Interview Panel
Award Date 2014-07-08T00:00:00+00:00
Financial Year 2013/14
Grant Programme: Title Investigator Award in Science
Internal ID 104634/Z/14/Z
Lead Applicant Dr Ariel Blocker
Partnership Value 750000
Planned Dates: End Date 2017-07-31T00:00:00+00:00
Planned Dates: Start Date 2015-03-16T00:00:00+00:00
Recipient Org: Country United Kingdom
Region South West