Coordinating Production and Distribution: the Pseudomonas aeruginosa ExsACDE Regulatory Cascade

Bacterial type III secretion systems (T3SSs) function to inject antihost proteins, termed effector proteins, directly into eukaryotic cells. Injected effector proteins disrupt host cell processes that normally function to limit the growth, multiplication, or distribution of a bacterial pathogen in its host. One hallmark of T3SSs is the use of complex regulatory circuits to coordinate effector protein expression with the activity of the T3S apparatus (4). Accordingly, activation of the T3S process triggers an almost simultaneous upregulation of effector gene transcription, while termination of the secretion process results in downregulation of effector gene transcription. The regulation of substrate expression in response to the activity of the T3S apparatus ensures substrate availability and efficient delivery of effector proteins. Interestingly, different T3SSs use different strategies to achieve this regulation, and although in general, the molecular details underlying these regulatory events are poorly understood, studies in the laboratory of Timothy Yahr have begun to provide an in-depth picture of one such regulatory system, the ExsADCE system of Pseudomonas aeruginosa (12). In this issue of Journal of Bacteriology, Brutinel et al. (2) use purified components to biochemically reconstitute the ExsADCE regulatory cascade in vitro. These studies confirm the previously established genetic model for this regulatory cascade of interacting proteins and provide novel insight into specific regulatory events. The primary transcriptional regulator of T3SS genes in P. aeruginosa is ExsA (1). ExsA is an AraC/XylS family protein that directly activates gene transcription by binding to 10 identified ExsA-dependent promoters (11). However, in the presence of calcium and prior to contact with a eukaryotic cell (conditions under which effector secretion is blocked), ExsA activity is inhibited by ExsD, an antiactivator that binds to the N-terminal domain of ExsA (6). Under these same conditions, ExsC, an anti-antiactivator and type III secretion chaperone, is bound to its cognate substrate ExsE (5, 7, 9, 10). The current model predicts that upon activation of the T3S process, ExsE is secreted, freeing ExsC to bind the antiactivator ExsD, dissociating the inactive ExsD/ExsA complex and liberating ExsA to bind ExsA-dependent promoters and activate transcription. Brutinel et al. (2) use purified ExsD/ExsA complexes, which exhibit no DNA binding activity in an electrophoretic mobility shift assay (8), to directly evaluate the ability of purified ExsC to bind ExsD, dissociate the ExsD/ExsA complex, and free ExsA to bind DNA. As predicted, the addition of a 10-fold molar excess of purified ExsC resulted in the dissociation of the ExsD/ExsA complex, formation of an ExsD/ExsC complex, and binding of ExsA to DNA. These important experiments clearly demonstrate that increased levels of unbound ExsC, as would be found upon secretion of ExsE, can directly dissociate the inactive ExsD/ExsA complex and release ExsA to bind DNA. Importantly, incubation of purified ExsC with excess ExsE, prior to adding ExsC to the ExsD/ExsA complex, prevented ExsC from dissociating the ExsD/ExsA complex. These studies confirm the current regulatory model and demonstrate that the ExsADCE regulatory cascade can be reconstituted in vitro with purified components. These studies further demonstrate that no additional factors are required for ExsC to dissociate the inactive ExsD/ExsA complex and release active ExsA. To verify that ExsD inhibits the ability of ExsA to bind DNA in vivo, Brutinel et al. (2) carried out chromatin immunoprecipitation assays with P. aeruginosa strains that either express or lack expression of ExsD under conditions where levels of ExsA were kept constant. The strains used for these studies also carried a lacZ transcriptional reporter for the ExsA-dependent exsD promoter (PexsD-lacZ). The presence of ExsD in the bacterial cell significantly reduced PexsD-lacZ reporter activity, as well as the ability of ExsA to be cross-linked to ExsA-dependent promoters in vivo, confirming that ExsD functions to prevent binding of ExsA to DNA and subsequent transcription of ExsA-dependent genes in vivo. Previous studies have demonstrated that ExsA binds to DNA as a monomer; however, once bound, ExsA also has the ability to recruit a second ExsA monomer through interactions mediated by the ExsA N-terminal domain (NTD) (1, 3). Brutinel et al. (2) confirm the ability of ExsA to dimerize via its NTD using a LexA-based one-hybrid system. Furthermore, they demonstrate that binding of ExsD to ExsA prevents this dimerization, indicating that ExsD prevents both ExsA DNA binding and multimerization. The role of ExsD's novel ability to block ExsA dimerization in its overall ability to inhibit ExsA function remains uncertain, as ExsD also blocks ExsA DNA binding and ExsA multimerization has previously only been observed following binding of ExsA to DNA. Nonetheless, it is possible that dimerization of ExsA plays a greater role in vivo than can be readily appreciated with the current in vitro assays. Indeed, the identification and analysis of ExsA mutants that specifically prevent dimerization but do not affect DNA binding will be required to fully determine the role of ExsA multimerization in the regulation of T3SS genes. Overall, the P. aeruginosa ExsADCE regulatory circuit represents a dynamic system that controls the expression of T3SS genes in response to the activity of the T3S apparatus. Interestingly, the activation of this system results in increased expression of essentially all T3SS genes, including those that assemble the T3S apparatus (12). Therefore, the initial activation of this regulatory cascade upon contact with host cells likely results in the assembly of increased numbers of functional T3S injectisomes (in addition to increasing substrate/chaperone expression). Thus, this system may also function as a type of early warning system that functions to better arm the bacterium for future encounters with host cells.