Structure and molecular function of the pseudopilus in type 2 protein secretion pathway – SYNERGY_T2SS

The goal of this project is to determine the molecular mechanisms that couples pseudopilus assembly with protein transport in T2SS. Recent advances in structural analysis of pseudopilus components and secreted exoproteins in partner teams makes this ambitious goal attainable. By forming the SYNERGY_T2SS consortium, six partners will build on their highly complementary expertise to dissect T2SS mechanism at the molecular level. Three experts in T2SS biology in different human (P1, P2) and plant pathogens (P3) will join forces with top level structural biologists, experts for NMR and biochemical analysis (P4), structural modeling (P5) and cryo-electron microscopy (P6).
In a systematic and concerted effort, we will determine, for the three T2SS models, the structure of the native T2SS pseudopilus composed of the core (task 1) and the tip complex (Task2), its interactions with substrates (task 3) and its in vivo and in silico dynamics (task 4). We will determine how pseudopilus assembly promotes protein secretion in T2SS at the atomic level, by focusing on structural, biophysical and functional analyses of pseudopilus interactions with selected substrates in vitro and in vivo. These are key objectives to understanding the molecular basis of substrate specificity in T2SS and the unique pseudopilus-mediated secretion mechanism. Furthermore, we will characterize pseudopilus dynamics in vivo and in silico. Recent advances in pilin fluorescent labeling using maleimide-coupled fluorophores will be used to track pseudopilus assembly dynamics by live fluorescence microscopy in model bacteria. This approach will allow us to determine whether pseudopilus retracts or permanently assembles in vivo, following a treadmill dynamics.

Despite the covid crisis, progress has been made in the tasks planned for the first-year period. Some of the project’s results have already been published three original mono-partner articles and one multi-partner review article.
The work of P2 on the Xcp pseudopilus structure in Pseudomonas aeruginosa led to the first atomic model of the complete T2SS pseudopilus. This model shows an extension of each of the four protofilaments in the pseudopilus core (already resolved by P1, P4 and P5) by one of the four minor pseudopilins, which are organized in a quaternary complex localized at the pseudopilus tip. This model fits perfectly in the secretin central cavity through which pseudopilus most likely transits to the cell surface. The publication describing these results is currently in revision in the journal Structure and has been deposited as a preprint available in BioRxiv server (doi.org/10.1101/2020.12.11.420943).

P3 published the results that highlight the molecular mechanisms of substrate recognition in the T2SS (doi.org/10.1016/j.jbc.2021.100305). Structure function analysis of secretion signals carried by exoproteins of Dickeya and Pectobacterium showed that, instead of a unique structural element, several distant and structurally flexible regions act together as a composite secretion signal in these proteins. We showed that one of these key regions undergoes disorder to order transitions, which could reflect its transient structuring during substrate recruitment by the T2SS. This type of dynamic behavior could be generally relevant for substrate recognition mechanism.
P1 participated in a collaborative study showing the presence of a T2SS in the mitochondria of several species of primitive unicellular organisms of the order Excavata. This study suggests an important role that T2SS might have played in these organelles close to the ancestral endosymbiont bacteria.

Fundamental insight in the secretion mechanism should allow us to intelligently harness T2SS for applications in vaccinology,
antimicrobial therapy, synthetic biology and bioremediation. Providing a unifying model of type 2 secretion will open up possibilities of manipulating the T2SS nanomachine to create desired substrates de novo.

1. Structure and function of minor pilins of type IV pili.
Jacobsen T, Bardiaux B, Francetic O, Izadi-Pruneyre N, Nilges M. Med Microbiol Immunol. 2020 Jun;209(3):301-308. doi: 10.1007/s00430-019-00642-5.(P1, P4, P5)

2. Pineau, C., N.Guschinskaya, IR Gonc¸alves, F. Ruaudel, X. Robert, P.Gouet, L. Ballut, VE Shevchik . Structure–function analysis of pectate lyase Pel3 reveals essential facets of protein recognition by the bacterial type 2 secretion system. J Biol Chem. 2021 Jan 16;100305. doi: 10.1016/ j.jbc.2021.100305. (P3)

3. Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system.« by L. Horváthová, V. Žárský, T. Pánek, R. Derelle, J. Pyrih, Al. Motycková, V. Klápštová, M. Vinopalová, L. Markova, L. Voleman, V. Klimeš, M. Petru, Z. Vaitová, I. Cepicka, K. Hryzáková, K. Harant, Mi. Gray, M. Chami, I. Guilvout, O. Francetic, B. F. Lang, C. Vlcek, A. Tsaousis, M. Elias, and P.Dolezal [Paper in press #NCOMMS-18-17107D. (P1)

4. Multidisciplinary Interrogation of a Crucial Protein Interface in the Type II Secretion System Escobar CA, Douzi B, G. Ball, B Barbat, S.A. Alphonse, L. Quinton, R. Voulhoux, KT Forest 2020.12.11.420943; BioRxiv preprint. doi: doi.org/10.1101/2020.12.11.420943. (P2)

Protein secretion systems are key determinants that contribute to bacterial ability to acquire nutrients, transform or colonize their niche and affect health of plants, animals and humans. The type II secretion system (T2SS) is a nanomachine composed of 12 essential protein components, forming a giant Mega Dalton complex that spans the two membranes of gram negative bacteria. T2SS promotes secretion of native, fully folded proteins and preassembled protein complexes that function as toxins, hydrolytic enzymes, adhesins or cytochromes. T2SS substrates (exoproteins) are recognized and secreted through a unique mechanism that requires assembly of a pilus-like structure called the pseudopilus. Secretion involves specific molecular recognition of structural determinants present on the surface of the pseudopilus and of the secreted cargo. Despite decades of study, the molecular determinants and steps of this highly specific and efficient transport have remained elusive.
Two contrasting continuous and discontinuous models have been proposed for the T2SS mechanism. In the continuous (Archimedes’ screw) model, pseudopilus is assembled at the inner membrane base and degraded at its tip, maintaining constant length like a molecular treadmill. In this model, substrates bind to the fiber core composed of the major pseudopilin GspG whose continuous assembly carries the cargo out of the cell. The discontinuous or piston-like model predicts that substrates interact with the pseudopilus tip complex, composed of four minor pseudopilins GspH-I-J-K. In this model, pseudopilus extension, which drives exoproteins across the outer membrane to the extracellular milieu, is followed by retraction that resets the system and allows reloading of new cargo in the periplasm.
The goal of this project is to determine the molecular mechanisms that couples pseudopilus assembly with protein transport in T2SS. Recent advances in structural analysis of pseudopilus components and secreted exoproteins in partner teams makes this ambitious goal attainable. By forming the SYNERGY_T2SS consortium, six partners will build on their highly complementary expertise to dissect T2SS mechanism at the molecular level. Three experts in T2SS biology in different human (P1, P2) and plant pathogens (P3) will join forces with top level structural biologists, experts for NMR and biochemical analysis (P4), structural modeling (P5) and cryo-electron microscopy (P6).
In a systematic and concerted effort, we will determine, for the three T2SS models, the structure of the native T2SS pseudopilus composed of the core (task 1) and the tip complex (Task2), its interactions with substrates (task 3) and its in vivo and in silico dynamics (task 4). We will determine how pseudopilus assembly promotes protein secretion in T2SS at the atomic level, by focusing on structural, biophysical and functional analyses of pseudopilus interactions with selected substrates in vitro and in vivo. These are key objectives to understanding the molecular basis of substrate specificity in T2SS and the unique pseudopilus-mediated secretion mechanism. Furthermore, we will characterize pseudopilus dynamics in vivo and in silico. Recent advances in pilin fluorescent labeling using maleimide-coupled fluorophores will be used to track pseudopilus assembly dynamics by live fluorescence microscopy in model bacteria. This approach will allow us to determine whether pseudopilus retracts or permanently assembles in vivo, following a treadmill dynamics.
Fundamental insight in the secretion mechanism should allow us to intelligently harness T2SS for applications in vaccinology, antimicrobial therapy, synthetic biology and bioremediation. Providing a unifying model of type 2 secretion will open up possibilities of manipulating the T2SS nanomachine to create desired substrates de novo.