Molecular mechanism of Type III secretion systems
PI: Åke Forsberg, Professor
Department of Molecular Biology
T3SSs are organized into supra-molecular structures known as needle complexes and this structure, which is believed to be common to all T3SSs, resembles a syringe with a base structure and a needle-like protrusion extending from the surface of the pathogen. The entire needle complex is traversed by a fine, hollow tube that may allow passage of effector proteins across the bacterial cell envelope. Due to the suggestive similarities between the needle complex and a syringe, it has been generally accepted that the effectors travel directly through the needle complex from the bacterial cytosol into the eukaryotic target cell through a pore induced by the secreted translocaters. Recent studies that have shown that both the effectors and translocaters actually are present on the surface of the bacterial cell prior to cell contact and that purified effectors added externally to an infected can be targeted into the host cell. Based on this an alternative working model for translocation has been suggested. (Figure 1 below).
Immunoelectron-micrograph showing localization of YopE of Yersinia pseudotuberculosis during infection of HeLa cells. Arrows indicate YopE detected on the surface or within the plasma membrane (pseudocolored red) of the target cell and stars (*) indicate YopE on the bacterial envelope.
Image courtesy of Roland Rosqvist, Lenore Johansson, the Electron Microscopy Platform at the Chemical Centre, Umeå University, Sweden.
In our ongoing research we are using genetics to introduce different fluorescence and epitope tags in genes encoding the different T3SS components, regulators and secretion substrates to facilitate studies of the kinetics and mechanism of T3SS mediated targeting of virulence effectors into host cells using advance fluorescence and electron microscopy. The microscopy studies are conducted at the Biochemical Imaging Centre Umeå (http://www.kbc.umu.se/platforms/bicu.html) and Umeå Core Facility Electron Microscopy (http://www.kbc.umu.se/platforms/ucem.html).
Figure1: Proposed model for T3SS-dependent protein translocation by a binary AB toxin like mechanism. T3SS translocators (t) and effectors (e) are secreted by the T3SS across the bacterial envelope (IM and OM) to the surface of the cell before host cell contact (1). Target cell sensing results in release of the surface localized T3SS substrates (2). The translocators (t) assemble into a pore in the target cell plasma membrane (PM) and mediate the translocation of the effectors (e) into the target cell cytoplasm (3).
Akopyan K, Edgren T, Wang-Edgren H, Rosqvist R, Fahlgren A, Wolf-Watz H, Fällman M (2011) Translocation of surface-localized effectors in type III secretion. Proc Natl Acad Sci U S A 108(4): 1639-1644.
Edgren T, Forsberg Å, Rosqvist R, Wolf-Watz H (2012) Type III secretion in Yersinia: injectisome or not? PLoS Pathog 8(5): e1002669.
Tat secretion in Yersinia and Pseudomonas
I. Tat-substrates in Yersinia.
In a previous study we could establish that Tat-secretion is essential for in vivo virulence of Yersinia pseudotuberculosis. Using a recently published improved bioinformatic approach we could identify approximately 30 potential Tat-substrates in Yersinia. One of the aims of this work is to verify which of these candidate proteins that are actually secreted by Tat system in Yersinia. The second major aim is to identify the Tat substrates that are responsible for the major attenuation of Tat mutants in the animal infection model.
II. Tat-substrates and screening for inhibitors in Pseudomonas
The bioinformatic approach identified at least 50 putative Tat- substrates in Pseudomonas. Several virulence factors or mechanisms including pyoverdin (iron chelater), phosholipase toxins and proteases have been implicated to be secreted via Tat in Pseudomonas. Previous work by other groups has verified that a tatC mutant in Pseudomonas is attenuated for virulence.
In addition, we have identified several novel potential Tat substrates including PelA that is required for biofilm formation and which is of great importance for survival of Pseudomonas in very harsh environments. We are currently performing screenings of chemical libraries at Laboratories for Chemical Biology Umeå (LCBU) at UmU. Specific tatC mutants will be included as negative controls in the HTS assay. Candidate Tat inhibitor compounds will be validated in dose response experiments and the best candidates will be further optimized by modeling and chemical synthesis provided by at LCBU.
Ochsner, U. A., A. Snyder, A. I. Vasil, and M. L. Vasil. 2002. Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis. Proc. Natl. Acad. Sci. USA 99:8312–8317.
Lavander, M, Ericsson, SK, Bröms, JB and Forsberg, Å. 2006. The twin arginine translocation system is essential for virulence of Yersinia pseudotuberculosis. Infect Immun 74:1768-76.
III. Structural/functional studies of prepilin peptidases
Prepilin peptidases have a unique role in that they are absolutely essential for the function of two highly conserved virulence mechanisms, type II secretion systems (T2SS) and type IV pili (Tfp). T2SS and Tfp are important for virulence in many clinically relevant bacterial pathogens. In collaboration with Pontus Gourdon, Copenhagen University and Mikael Elofsson, LCBU, Umeå University we have initiated a new research project to structurally characterize the prepilin peptidase PilD of Psedomonas aeruginosa with the long term goal to evaluate this enzyme as a target for development of novel generic antimicrobials with limited selection pressure for resistance development.
Korotkov KV, Sandkvist M, Hol WG. 2012. The type II secretion system: biogenesis, molecular architecture and mechanism. Nat Rev Microbiol. 10:336
Salomonsson EN, Forslund AL, Forsberg Å. 2011. Type IV Pili in Francisella - A virulence trait in an intracellular pathogen. Front Microbiol 2: 29.