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

Abstract

Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to damage eukaryotic host cells and evade phagocytosis. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. ExsA-dependent transcription is coupled to type III secretory activity through a cascade of three interacting proteins (ExsC, ExsD, and ExsE). Genetic data suggest that ExsD functions as an antiactivator by preventing ExsA-dependent transcription, ExsC functions as an anti-antiactivator by binding to and inhibiting ExsD, and ExsE binds to and inhibits ExsC. T3SS gene expression is activated in response to low-calcium growth conditions or contact with host cells, both of which trigger secretion of ExsE. In the present study we reconstitute the T3SS regulatory cascade in vitro using purified components and find that the ExsD·ExsA complex lacks DNA binding activity. As predicted by the genetic data, ExsC addition dissociates the ExsD·ExsA complex through formation of an ExsD·ExsC complex, thereby releasing ExsA to bind T3SS promoters and activate transcription. Addition of ExsE to the purified system results in formation of the ExsE·ExsC complex and prevents ExsC from dissociating the ExsD·ExsA complex. Although purified ExsA is monomeric in solution, bacterial two-hybrid analyses demonstrate that ExsA can self-associate and that ExsD inhibits self-association of ExsA. Based on these data we propose a model in which ExsD regulates ExsA-dependent transcription by inhibiting the DNA-binding and self-association properties of ExsA.

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

“…At the broadest level, ExsA is a positive transcriptional regulator for T3SS1 genes 47 ( Fig. 11) and ExsA transcriptional activity is blocked when ExsD binds to ExsA 48 ( Fig. 11).…”

“…45,48 Under limited calcium conditions ExsD is bound by another protein, ExsC, and this blocks the ExsD-ExsA interaction and permits transcription of T3SS genes. 46 Although the conditions that are permissible for T3SS1 expression in V. parahaemolyticus probably do not mirror those required for T3SS expression in P. aeruginosa, the proximal means of controlling T3SS1 expression is very similar.…”

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…Transcription of T3SS-associated genes is intimately coupled to the state of the T3SS secretory apparatus by the small, T3SS-secreted regulatory factor ExsE (128). In the absence of inducing conditions (high Ca 2ϩ or host cell contact) the secretion channel is closed, (129)(130)(131)(132)(133)(134). Microarray experiments by Brencic et al determined that rsmA mutation reduces the expression of 22 T3SS-associated genes, including representatives from genes that facilitate secretion, translocation, regulation, and every exotoxin secreted by P. aeruginosa strain PAK (80,133).…”

Cheating by type 3 secretion system-negative Pseudomonas aeruginosa during pulmonary infection

“…ExsA, a member of the AraC/XylS family of transcriptional activators, activates its own transcription as well as that of a well-defined set of genes encoding T3SS apparatus proteins, chaperones, and effectors in response to host cell contact or in vitro inducing cues (e.g., calcium limitation) (5,6). ExsA activity is controlled by the anti-activator ExsD, which binds and inhibits ExsA when T3SS-inducing cues are absent (7). ExsD repression is relieved when inducing cues trigger secretion of the T3SS substrate ExsE, thereby freeing its binding partner ExsC to interact with ExsD.…”