Characterization of ExsC and ExsD self-association and heterocomplex formation.

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced by calcium depletion and is positively regulated by the ExsA transcriptional activator and negatively regulated by the ExsD antiactivator. Under conditions permissive for expression of the T3SS, the negative regulatory activity of ExsD is antagonized by a direct binding interaction with ExsC. In the present study, the ExsC-ExsD binding interaction was characterized. Individually, both ExsC and ExsD form self-associated complexes, as judged by bacterial monohybrid and gel filtration experiments. A mixture of purified ExsC and ExsD readily formed a complex that elutes from gel filtration medium as a single included peak. The calculated molecular weight of the ExsC-ExsD complex is consistent with a complex containing multiple copies of ExsC and ExsD. Isothermic titration calorimetry experiments found formation of the ExsC-ExsD complex to be thermodynamically favorable, with a Kd of approximately 18 nM and a likely binding ratio of 1:1. To identify amino acid residues important for the regulatory activities of ExsC and ExsD, self-association, and complex formation, charged-cluster mutagenesis was performed. Two of the resulting ExsD charged-cluster mutants (DM2 and DM3) demonstrated a hyperrepressive phenotype for expression of the T3SS. By two-hybrid and copurification assays, the DM3 mutant was found to be impaired in its interaction with ExsC. This finding demonstrates that the binding of ExsC to ExsD is required for transcriptional induction of the T3SS under calcium-limiting growth conditions.

A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence determinant. Transcription of the T3SS is highly regulated and intimately coupled to the activity of the type III secretion channel. The secretion channel is generally closed, and transcription is repressed. Inducing signals such as calcium depletion, however, open the secretion channel and derepress transcription of the T3SS. The coupling of transcription with secretion requires three previously identified cytoplasmic regulatory proteins. ExsA is a DNA-binding protein required for transcriptional activation of the entire T3SS. The second regulatory protein, ExsD, functions as anti-activator by directly binding to ExsA. Finally, ExsC functions as an anti-anti-activator by directly binding to and inhibiting ExsD. Although the regulatory roles of ExsC, ExsD, and ExsA were defined through these previous studies, the mechanism of coupling transcription to secretion was unclear. We now report the identification of ExsE as a secreted regulator of the T3SS and provide evidence that ExsE functions as a direct inhibitor of ExsC. When the secretion channel is closed, ExsE is complexed with ExsC in the cytoplasm, and transcription of the T3SS is repressed by sequestration of ExsA by ExsD. We propose that the secretion of ExsE provides an initiating signal that results in an equilibrium shift whereby ExsC becomes preferentially bound to ExsD, thus allowing liberated ExsA to activate transcription of the T3SS. The presence of ExsE homologs in the T3SSs of other bacterial species suggests that this mechanism of coupling transcription to secretion may be commonly used.

A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system.

Expression of the Pseudomonas aeruginosa type III secretion system (TTSS) is coupled to the secretion status of the cells. Environmental signals such as calcium depletion activate the type III secretion channel and, as a consequence, type III gene transcription is derepressed. Two proteins, ExsA and ExsD, were shown previously to play a role in coupling transcription to secretion. ExsA is an activator of TTSS gene transcription, and ExsD is an anti-activator of ExsA. In the absence of environmental secretion cues, ExsD binds ExsA and inhibits transcription. Here, we describe the characterization of ExsC as an anti-anti-activator of TTSS expression. Transcription of the TTSS is repressed in an exsC mutant and is derepressed upon ExsC overexpression. The dependence on exsC for transcription is relieved in the absence of exsD, suggesting that ExsC and ExsD function together to regulate transcription. Consistent with this idea, ExsC interacts with ExsD in bacterial two-hybrid and co-purification assays. We propose a model in which the anti-anti-activator (ExsC) binds to and sequesters the anti-activator (ExsD) under low Ca(2+) conditions, freeing ExsA and allowing for transcription of the TTSS. The P. aeruginosa system represents the first example of an anti-activator/anti-anti-activator pair controlling transcription of a TTSS.

ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon.

Expression of the Pseudomonas aeruginosa type III secretion system is induced by contact with eukaryotic cells, serum or low Ca2+ concentrations. We report that ExsD, a unique protein, is a negative regulator of the type III regulon. Localization studies indicate that ExsD is not secreted by P. aeruginosa. To determine the role of exsD, a non-polar deletion was returned to the chromosome by allelic exchange. The delta exsD mutant is competent for type III secretion and translocation of the ExoU cytotoxin to eukaryotic host cells. To examine the effect of ExsD on transcription, lacZ transcriptional reporter fusions were integrated into the chromosome. Promoters controlling transcription of genes encoding the type III secretory, regulatory and effector proteins demonstrated significant derepression in the delta exsD background. Expression of ExsD from a multicopy plasmid completely repressed transcription of the regulon. Although a mutant in pscC, encoding a structural component of the type III translocase, is repressed for expression of the regulon, a delta exsD, pscC:: omega double mutant is derepressed. Bacterial two-hybrid data indicate that ExsD binds the transcriptional activator of the regulon, ExsA. We conclude that ExsD is a negative regulator and propose that ExsD functions as an ExsA antiactivator to regulate transcription of the regulon.