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

Abstract

SummaryExpression 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 antiactivator 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-antiactivator (ExsC) binds to and sequesters the antiactivator (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.

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

“…43 ExsD binds ExsA directly and this blocks ExsA transcriptional activity 44,45 while ExsC binds ExsD to permit ExsA transcriptional activity. 43,46 that differences in band intensities for different conditions were not due to unequal protein loading on the gels (Fig. 2, lower).…”

“…vp1701 is located proximal to the T3SS1 transcriptional regulator exsA (vp1699) and the protein shares 34% amino acid identity with ExsC found in Pseudomonas. 46 ExsC regulates expression of T3SS in Pseudomonas by interacting with ExsD and thus the homologous protein in V. parahaemolyticus may have a similar function. We infected HeLa cells or Caco-2 cells with V. parahaemolyticus strain NY-4 (wild-type) and a ∆exsC strain (exsC deletion mutant) and 4 hr after infection we collected the whole-cell lysates to examine the synthesis of two T3SS1 substrates (Vp1656 and Vp1659).…”

Control of gene expression by type III secretory activity

“…2A) [29]. ExsC functions as an anti-anti-activator by binding to and inhibiting the negative regulatory activity of ExsD [30]. ExsC also serves as a type III secretion chaperone for ExsE [31] [32].…”

Anti-activator ExsD Forms a 1:1 Complex with ExsA to Inhibit Transcription of Type III Secretion Operons

“…The anti-activator ExsD belongs to the dedicated regulatory cascade that links synthesis of T3SS proteins to secretory activity (21). This pathway involves also the anti-anti-activator ExsC (23) and the secreted/ translocated protein ExsE (24,25). Based on in vivo and in vitro studies, a model of regulation has been proposed (10): when the T3SS is induced, after direct contact with the target cell or calcium depletion, the small protein ExsE is translocated through the syringe-like apparatus into the host cell; consequently, ExsC binds ExsD, which releases ExsA that becomes able to activate transcription of the T3SS genes.…”