Functional reconstitution of Type 3 protein targeting and secretion

Type III secretion (T3S) is a specialized protein export and delivery system for protein toxins. These effectors are directly injected in the cytoplasm of host eukaryotic cells, through a dedicated nanosyringe, the injectisome, that spans two bacterial and one eukaryotic membrane. Once secreted, effectors interfere with host signalling pathways and modulate cellular processes, leading to host cell death. The T3SS is widespread and essential for the pathogenic and symbiotic potential of many Gram- bacteria. It is responsible for several severe diseases, such as plague (Yersinia pestis), typhoid fever (Salmonella enterica) and infantile bacterial diarrhea (Enteropathogenic E. coli; EPEC) leading to millions of annual deaths. Despite significant progress towards understanding the molecular basis of protein transport through the injectisome, the exact mechanism remains elusive.T3SS is one of the most complex protein secretion systems known. To become functional, up to 40 different proteins, in hundreds of copies, interact and assemble in a hierarchical manner. Despite sequence differences between different pathogenic and flagellar T3SS subunits, all core components share significant structural homology, indicating ancestry. Therefore, protein function in one bacterium is commonly deduced, by homology and/or biochemical similarity to that in other species.Assembly and secretion through the T3SS is stepwise, hierarchical and finely tuned. The system ensures that structural components are secreted first to build the injectisome, followed by the effectors. Assembly of the T3SS apparatus occurs in distinct steps: a) basal body formation, b) formation of the needle and translocators by secretion of early and middle substrates, respectively, c) switch from translocators to effectors (late substrates) and injection of the latter in the host cytoplasm. During middle to late substrate switching, the gatekeeper SctW regulates membrane targeting of chaperone/secretory protein complexes to SctV. SctV-bound SctW increases the binding affinity of the early and suppresses that of late substrates for SctV. Conformational changes in the cytoplasmic domain of SctV might regulate protein sorting and targeting, yet this hypothesis remains untested.The positioning of the ATPase complex/C-ring at the cytoplasmic interface of the export apparatus has led to the hypothesis that these components are essential for protein sorting/targeting . However, these peripheral components are easily detached from the membrane, their association is quite dynamic and are not essential for in vitro targeting of secretory proteins. Therefore, the role of the ATPase complex/C-ring in sorting needs to be clarified. The primary energy source for T3SS secretion is thought to derive from the proton motive force across the inner membrane, while ATP hydrolysis is believed to dissociate chaperone/secretory protein complexes. Nonetheless, the exact energizing mechanism required for protein translocation remains elusive.The detailed molecular mechanism by which proteins interact with the export apparatus so as to be delivered, enter the pathway and become secreted, the order of events and the interactions occurring to allow hierarchical secretion, as well as the high resolution structure of the apparatus are some of the fundamental questions that need to be addressed. Up to now deciphering this complex mechanism has relied mainly on in vivo genetic analysis, pull-down assays and high resolution structures of some of the components and/or of injectisomes in whole cell tomograms. Approaches used so far have some inherent limitations. In vivo phenotypic observation from single gene deletions, provides no information regarding intermediate steps of protein translocation. Trying to map protein interactions and determining affinities in solution using membrane protein domains and secretory protein fragments resulted in no interactions detected or 30-fold lower affinities compared to the ones probed using membrane vesicles with functional injectisomes. The lack of purified and reconstituted translocase is a critical obstacle. Monitoring protein translocation in vitro, is a major step towards improving our understanding of the fundamental aspects of T3SS function and the molecular basis of the system’s regulation. The complex and dynamic nature of interactions between T3SS components requires novel approaches to dissect the order of events during secretion. The proposed research combines cutting-edge structural dynamics methods, such as single molecule FRET (smFRET) and Hydrogen-deuterium Exchange Mass Spectrometry (HDX-MS), to decode the role of conformational dynamics of injectisome components, with high resolution structural analysis, in vitro functional assays and reconstitution of the protein release mechanism.The scientific research objective is to addresses T3SS protein sorting, targeting and secretion. Taking advantage of existing knowledge on the T3SS pathway, analysis of secretion pathways in biochemical and biophysical will be done with three main objectives: a. Structural and functional characterization of the SctV receptor: 1) establish in vitro assays to monitor SctV interaction with soluble T3S protein/or chaperone/secretory protein complexes;2) determine the structure of nonameric-SctV;3) global structural dynamics of Peptidisc-reconstituted SctV by HDX-MS;4) determine conformational changes and allosteric events that occur on SctV upon interaction with chaperone/secretory protein complexes.b. Structural and functional characterization of the holo-export apparatus SctRSTUV:1) reconstitute a stable holo-export apparatus (SctRSTUV) in vitro;2) establish an in vitro assay to monitor T3S protein binding on the reconstituted export apparatus;3) determine the structure of the holo-export apparatus.c. Reconstitution of delivery/release of secretory proteins to the export apparatus in vitro. 1) biochemical characterization of assembly of T3SS cytoplasmic components on the holo-export apparatus;2) mapping the exported protein mode of SctV crossing and release.And the established and new tools are used in vitro assays to: a. deconvolute the order of events during protein secretion through the injectisome;b. decode the role of the conformational dynamics of the export apparatus.Clarifying those will be a major step towards understanding the system’s regulation. And several biomedical/biotechnological anticipated applications are: a. improved understanding of bacterial infection diseases; b. recombinant vaccines derived from T3SS-exported antigens;c. novel assays for anti-virulence compounds that block essential biochemical steps identified here in collaboration with the in-house drug development company CD3 at KUL.During the research, FReT3 will be materialized by combining biochemical and biophysical tools, HDX-MS, smFRET and high resolution EM so as to acquire detailed functional, structural and dynamic insights on T3SS protein targeting and secretion.a. SctV will be structurally and functionally characterized in vitro via an intercontinental-collaborative network. Peptidisc technology is a universal, high-throughput, biochemical approach for keeping membrane-protein complexes stably in-solution, avoiding incompatible buffers for biophysical methods, such as detergents and artificial lipid bilayers. Peptidisc reconstitution will be the pipeline used to proceed to determining the structure of SctV by Cryo-EM, in collaboration with the Marlovits lab (CSSB, Hamburg).Furthermore, the receptor function of SctV will be analysed, using purified T3SS chaperone/secretory protein complexes and biophysical tools, such as Isothermal Titration Calorimetry (ITC) (for affinity measurements and thermodynamic parameters) and Size-Exclusion Chromatography in line with Multi laser Angle light scattering (SEC-MALS) and native Mass Spectrometry (for complex formation stoichiometries).b. The structural dynamics of Peptidisc-reconstituted SctV, will be studied by HDX-MS, which is uniquely powerful in providing detailed information of conformational flexibility and dynamics down to near-residue level. The established HDX-MS protocols which has been extended to characterizing membrane embedded Sec protein complexes will be applied to the T3SS to determine the global structural dynamics of SctV on its own and in the presence of different substrates, such as the gatekeeper SctW, the chaperone/secretory protein complex CesT/Tir or CesF/EspF that bind to SctV with nanomolar affinity.Additionally, smFRET monitors intra- and inter-molecular dynamics and probes conformational alterations in a protein, by calculating FRET efficiencies of a donor-acceptor fluorophore pair as a function of time and converting them to an inter-probe distance. This complementary biophysical technique will be used to follow the different conformational states of each SctV protomer, within the nonameric context, as secretory proteins bind to SctV. Conformational changes monitored by smFRET will be probed using in solution confocal microscopy using the MIcrotime200 (Picoquant). While HDX-MS will follow the average D uptake of a peptide from all the protomers of the SctV nonamer and monitor intramolecular changes, smFRET will monitor conformational dynamics in a single protomer of the nonamer.c. Reconstitute the complete T3SS export apparatus in vitro. The T3SS export apparatus comprises five inner membrane proteins SctR/S/T/U/V in EPEC. All five genes have already been cloned in different expression vectors separately or in operons. A variety of heterologous expression strains will be tested so as to maximize protein synthesis and purification will be performed using mild, non-ionic detergents, as for SctV alone. Once the protein complex is reconstituted in vitro, the pipeline established above (see “a”) will be followed for structural and functional characterization using Cryo-EM and biochemical approaches.d. An assay to monitor T3S translocation in vitro using IMVs and purified chaperone/secretory protein complexes has been set up. Using radiolabelled exported proteins we monitor protein release into the lumen of the vehicles. This reaction will be coupled to biochemical activities such as monitoring of ATP hydrolysis by the ATPase of the system that will be provided in trans. To determine the assembly requirements of the complete set of peripheral components, we have generated genetic constructs that allow over-expression and purification of the components alone or in combinations. All purified soluble complexes will be primarily characterized with SEC-MALS and ITC for complex formation determination and affinities. Additionally, using live cell imaging, cryoEM and in vitro translocation assays, we will monitor secretory protein release in the lumen of IMVs and resolve the structural dynamics of the export apparatus during the process.

Keywords:T3SS, secretory proteins, functional reconstitution

Disciplines:Infectious diseases, Bacteriology, Cellular interactions and extracellular matrix, Molecular physiology, Structural biology

Project type:PhD project