Characterization of ExsC and ExsD Self-Association and Heterocomplex Formation

Abstract

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 K d of ϳ18 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 chargedcluster 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.

ExsD Inhibits Expression of thePseudomonas aeruginosaType III Secretion System by Disrupting ExsA Self-Association and DNA Binding Activity

“…For this reason ExsD was coexpressed with either ExsC His or ExsA His from the dual expression vector pCOLADuet-1 in E. coli and purified by Ni 2ϩ affinity chromatography. Both the purified ExsD·ExsC His and ExsD·ExsA His complexes were soluble based on resistance to high-speed sedimentation (100,000 ϫ g for 30 min), and both eluted from gel filtration columns as single included peaks with apparent molecular masses of 186 and 97 kDa, respectively (data not shown), which is consistent with previous studies (20,28).…”

Regulation of type III secretion system 1 gene expression inVibrio parahaemolyticusis dependent on interactions between ExsA, ExsC, and ExsD

“…By substitution of charged residues with alanine or glycine, Lykken et al 49 identified several ExsC mutants that were not able to complement the phenotype of exsC mutant. Nevertheless, these mutants were still able to bind ExsD, suggesting that binding of ExsC with ExsD is not sufficient for the induction of T3SS in Pseudomonas.…”

ExsA and LcrF Recognize Similar Consensus Binding Sites, but Differences in Their Oligomeric State Influence Interactions with Promoter DNA

“…ExsD functions as an antiactivator by directly binding to the amino-terminal domain (NTD) of ExsA and inhibiting ExsA-dependent transcription (9,10). ExsC is an anti-antiactivator that binds to and antagonizes the inhibitory activity of ExsD (11)(12)(13). The final component of the cascade, ExsE, is a secreted substrate of the type III export machinery.…”