Type III Secretion in Yersinia: Injectisome or Not?

Wolf-Watz H (2012) Type III Secretion in Yersinia: Injectisome or Not? PLoS Pathog 8(5): e1002669. doi:10.1371/ journal.ppat.1002669 Type III Secretion in Yersinia : Injectisome or Not? Tomas Edgren 0 1 A ke Forsberg 0 1 Roland Rosqvist 0 1 Hans Wolf-Watz 0 1 The Ca 0 1 Paradox 0 1 Virginia Miller, University of North Carolina at Chapel Hill, United States of America 0 Funding: This work was supported by the Swedish Research Council (VR), the Kempe Foundation, the Carl Trygger Foundation for Scientific Research, and the Foundation for Medical Research (Insamlingsstiftelsen) at Umea University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 1 Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea Centre for Microbial Research (UCMR), Umea University, Umea , Sweden, 2 The Electron Microscopy Platform at KBC, Umea University , Umea , Sweden Human pathogenic Yersinia species are uniquely dependent on millimolar concentrations of Ca2+ when grown at 37uC. This phenotype is characterized by normal growth at 37uC in the presence of Ca2+, and growth restriction in Ca2+-depleted medium [1]. This unusual Ca2+ requirement has been linked to the presence of a virulence plasmid [2], originally discovered by Zink et al. [3]. The plasmid encoded a set of proteins (Yersinia outer proteins, or Yops) [4] that were secreted into the culture supernatant in massive amounts during growth restriction in the absence of Ca2+ [5,6]. The coupling of the Yops to the virulence plasmid indicated strongly that they were essential virulence determinants. This hypothesis was further supported when convalescence sera obtained from Yersinia-infected patients were shown to contain antibodies recognizing the secreted Yop proteins [4], leading to the hypothesis that low intracellular Ca2+ inside mammalian cells induced expression of the Yop virulon during infection. The role of calcium was thus paradoxical as conditions promoting Yop secretion resulted in growth restriction. - Under certain conditions, pathogens like Yersinia secrete high levels of proteins. This ability correlates with cytotoxicity and virulence. The complete inactivity of the secreted proteins when added directly to host cells stimulated research to disclose this non-AB toxin mechanism of virulence, and eventually led to the discovery of the type III secretion mechanism and the injection model for virulence effector targeting. Recent studies question this model and suggest that the T3SS effector-targeting mechanism may resemble the classical AB toxin delivery mechanism. Figure 1. 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). doi:10.1371/journal.ppat.1002669.g001 passage of effector proteins across the bacterial cell envelope [19]. 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 lumen of the eukaryotic target cell. However, no experimental results have been presented to demonstrate that the effectors are secreted through the needle structure. Are the Effectors Translocated through a Pore Formed in the Host Cell Membrane? The secreted substrates can be divided into two functional classes: effectors are delivered into the target cell where they elicit a biological response, while translocators facilitate the delivery of effectors across the plasma membrane [10,12,13]. A key signature of translocators is the putative membrane spanning domain(s), which has some similarities to pore-forming toxins [20]. The first translocator protein found to exhibit pore-forming and hemolytic activities was Shigella IpaB [21]. Subsequently, the Yersinia homologue YopB was found to be essential for Yop effector translocation; similar to IpaB, YopB also induces erythrocyte hemolysis [22,23]. The pore-forming ability of YopB correlates with functional translocation, supporting the idea that the effectors are delivered through a pore in the host cell membrane. Recent work has revealed that LcrV (initially thought to be a translocator protein) is localized at the tip of the needle complex [24]. LcrV has also been proposed to be essential for insertion of the hydrophobic translocators YopB and YopD into target cell membranes. These properties of LcrV agree nicely with the injection model. However, all findings are not compatible with this model. For example, Sasakawa and co-workers reported that the Shigella translocators IpaB, IpaC, and IpaD are surface-localized before target cell contact [25]. The majority of these proteins are rapidly released after target cell contact is established, indicating that Shigella senses target cell contact and responds accordingly. Furthermore, latex beads coated with purified Ipa protein complexes are internalized by target cells through a mechanism that resembles the active, T3SS-dependent engulfment of Shigella [26]. The apparent surface localization of these proteins before target cell contact is difficult to reconcile with the injection model. On the other hand, recent studies in Shigella demonstrating that IpaB and subsequently IpaC are recruited to the tip of the needle from its location at the bacterial surface in response to target cell contact lend support to the injection model [27]. Nevertheless, these results do not support the idea that the T3SS forms a conduit that allows both translocators and effectors to be secreted in one step by the same T3SS, since at least the translocators are secreted to the surface before eukaryotic cell contact has been established. Binary AB Toxin Revisited? Recent attempts to visualize T3SS substrates during infection using immunogold labeling and transmission electron microscopic analysis revealed that the majority of Yop translocators and effectors are present on the surface of the bacterium before target cell contact [28]. This finding was surprising because the injection model predicts that the T3SS substrates should be present in a secretion competent conformation in the bacterial cytosol before target cell contact. Remarkably, a yopH null mutant coated with purified YopH was translocation competent and complemented the mutant phenotype. The translocation of surface-localized YopH and YopH-b-lactamase reporters is also T3SS dependent and requires functional translocators, as well as a specific translocation domain present in YopH. T (...truncated)