Limiting too much of a good thing: a negative feedback mechanism prevents unregulated translocation of type III effector proteins.

Type III secretion systems (T3SS) are highly specialized structures termed injectisomes which function by translocating bacterial effector proteins via hollow needles directly into eukaryotic target cells. The translocated effectors alter target cell physiology to promote the pathogenic and symbiotic lifestyles of many gram-negative bacterial species. Orchestrating T3SS activity requires multiple levels of regulatory control to ensure appropriate timing of gene expression, needle complex assembly, pore formation in the target cell membrane, and translocation of the effectors into target cells. Most of the work thus far has focused on regulatory events leading up to and including translocation of the effectors. Although fewer studies have examined regulatory events that occur following translocation of the effectors into target cells, recent evidence indicates that translocation in Yersinia pseudotuberculosis is a regulated event that is controlled by the translocated YopE effector (1). In this issue of the Journal of Bacteriology, Cisz et al. demonstrate that a similar negative feedback mechanism operates in Pseudomonas aeruginosa (3). Using an elegant microscopy-based assay, they provide evidence that the autolimiting effect on translocation prevents subsequent bacteria from deploying the T3SS against the same target cell. In a separate line of investigation, they propose that host contact-mediated activation of translocation involves a shift in the substrate specificity of the injectisome and provide evidence that the signal associated with target cell contact is not the low-Ca environment of the host cell cytoplasm. Arming and subsequent activation of the T3SS. The P. aeruginosa T3SS elicits cytotoxicity toward eukaryotic target cells through the activity of four translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) (2, 13, 18). Translocation of the effectors is dependent upon 36 genes that encode proteins comprising the needle complex, translocators, chaperones, and regulators (5). These genes are transcribed at a basal level in the absence of inducing signals, allowing for assembly of one or two needle complexes per cell (11). As a result, P. aeruginosa is preloaded with small amounts of the effector proteins and is poised to rapidly deploy the T3SS. The T3SS remains in standby mode until activation signals trigger secretion and/or translocation of the effector and regulatory proteins and up-regulation of T3SS gene expression. The primary activating signals are growth under Ca -limiting conditions or contact of P. aeruginosa with target cells (17). Activation by the low-Ca signal was previously thought to trigger secretion of the effector proteins as well as the translocators (PcrV, PopB, and PopD) and a negative regulator of T3SS gene expression (ExsE) (9, 16). Secretion of ExsE into the growth medium or its translocation into target cells results in a concomitant decrease in the bacterial intracellular concentration of ExsE, thereby relieving the block on T3SS gene expression (16). The study by Cisz et al. presents evidence that whereas secretion of ExsE and the effector proteins is dependent upon the low-Ca signal, secretion of the translocator proteins (PcrV, PopB, and PopD) occurs irrespective of Ca levels (3). Thus, rather than generally activating the P. aeruginosa injectisome, low Ca triggers a switch in the substrate specificity of the injectisome. Interestingly, low Ca was recently shown to induce substrate switching of the enteropathogenic Escherichia coli injectisome as well (4). These important findings establish a hierarchy in secretion whereby translocator proteins are secreted first, allowing for rapid assembly of the translocation pore, followed by the regulated delivery of the effector proteins. It is worth noting, however, that translocator proteins secreted from one cell are unable to cross-complement translocator mutants in trans (3, 12). What then might be the purpose of constitutively secreting translocator proteins that are apparently unable to be recruited from the extracellular milieu and assembled into a functional pore complex? It is possible that formation of the translocation pore requires a high local concentration of the translocators and that these concentrations can be achieved only by constitutively secreting the translocators in the vicinity of the needle tip. An alternative explanation consistent with the data presented by Cisz et al. is that the translocators are stably associated with the needle complex near the cell surface. Rather than being continuously secreted, however, the translocators may be released into the culture medium by the shear forces associated with growth of P. aeruginosa under shaking conditions. Constitutive secretion of the translocators would then permit continuous regeneration of the needle complexes. A final possibility is that constitutive release of the translocators reflects an immune avoidance strategy. It is well established that antibody responses to one of the translocators (PcrV) prevent assembly of the trans* Corresponding author. Mailing address: University of Iowa, 540B Eckstein Medical Research Building, Iowa City, IA 52242-1101. Phone: (319) 335-9688. Fax: (319) 335-8228. E-mail: timothy-yahr @uiowa.edu. Published ahead of print on 14 December 2007.