A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system - PubMed (original) (raw)

Comparative Study

. 2005 Jul 12;102(28):9930-5.

doi: 10.1073/pnas.0504405102. Epub 2005 Jun 28.

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Comparative Study

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

Mark L Urbanowski et al. Proc Natl Acad Sci U S A. 2005.

Abstract

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.

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Figures

Fig. 1.

Fig. 1.

Proposed regulatory scheme for bacteria encoding ExsCDE homologs. (A) Model for regulation of the T3SS. Under high Ca2+ conditions, protein X (later identified as ExsE) remains cytoplasmic. This favors formation of the protein X·ExsC- and ExsD·ExsA-binding complexes (indicated by larger text and a larger dark circle) resulting in repression of the T3SS. Under low Ca2+ conditions, protein X is secreted. The decrease in intracellular protein X shifts the binding equilibrium in favor of forming the ExsC·ExsD complex and free ExsA. (B) exsE homologs are present in similar genetic contexts in other bacterial species. Comparison of the genetic organization of regions encoding putative homologs of the P. aeruginosa T3SS regulators ExsC, ExsA, and ExsD. The exsCEBA and the beginning of the exsD, pscB-L operons are indicated by arrows. Transcription of both of these operons is ExsA-dependent. The functions of ExsC, ExsA, and ExsD are indicated. ExsB is thought to be a chaperone involved in assembly of the PscC secretin and has no known role in transcriptional regulation. P.a., P. aeruginosa PA01; P.l., P. luminescens TT01; A.h., A. hydrophila. GenBank accession nos.: P.a., AE004091; P.l., BX571871; A.h., AY528667.

Fig. 2.

Fig. 2.

Characterization of type III secretion in the exsE deletion mutant. The indicated strains carrying the P_exsD_-lacZ reporter were grown under noninducing (–EGTA) or inducing (+EGTA) conditions. (A) β-Galactosidase activity (Miller units) from the P_exsD_-lacZ reporter. The reported values represent the average from three independent experiments. The standard deviation is indicated by the error bars. (B) Silver-stained gel of concentrated culture supernatants. The secreted effectors ExoU and ExoT are indicated, whereas the Pops represent the closely migrating proteins PopD, PopN, and PcrV. (C and D) Anti-ExoU immunoblot of concentrated culture supernatants (C) and whole-cell lysates (D).

Fig. 3.

Fig. 3.

The negative regulatory activity of ExsE depends on a functional ExsC/ExsD regulatory system. Strains carrying the P_exsD_-lacZ reporter were transformed with either a vector control (pJN) or ExsE (p_JexsE_), ExsD (pJ_exsD_), or ExsC (p_exsC_) expression vectors. The transformants were grown in the presence of the appropriate antibiotics under either noninducing (–EGTA) or inducing (+EGTA) conditions in the presence of 0.5% arabinose, and β-galactosidase activity (Miller units) was determined. The reported values represent the average from three independent experiments. When plotted on a log scale, some of the errors bars are not visible.

Fig. 4.

Fig. 4.

ExsE interacts with ExsC. (A) Two-hybrid interaction assay. The E. coli reporter strain SU202 carries plasmids expressing only the LexA or LexA408 DNA-binding domain (–) or fusions of the LexA or LexA408 DNA-binding domains fused to ExsE (E), ExsC (C), ExsD (D), or the control proteins Fos and Jun. Cells were grown in LB medium and assayed for β-galactosidase activity (Miller units). (B) ExsC copurification assay. A cell extract of E. coli overexpressing ExsC was mixed with either purified ExsEHis-6 (+) or with buffer (–); the mixtures were then subjected to Ni-NTA affinity chromatography. Aliquots of the mixture (load) and the column eluate were subjected to anti-ExsC and anti-His-6 immunoblot analyses. (C) ExsD copurification assay. A cell extract of E. coli overproducing ExsD was mixed with either purified ExsCHis-6 or purified ExsEHis-6 as indicated (+) or mixed with buffer (–). The mixtures were then subjected to Ni-NTA affinity chromatography and subjected to anti-ExsD and anti-His-6 immunoblot analyses.

Fig. 5.

Fig. 5.

ExsE is secreted in an ExsC-dependent response to low Ca2+. Wild-type PA103 and the indicated mutants carrying the p_exsE_His-6 expression plasmid were grown under either noninducing (–EGTA) or inducing (+EGTA) conditions for type III secretion in the presence of 0.5% arabinose. Culture supernatants (sup) (derived from 4.5 × 108 cells), and cell-associated soluble and insoluble (insol) fractions (derived from 1.15 × 108 cells) were immunoblotted with anti-His-6. Lanes A1–6, PA103; lanes B1–6, Δ_exsD_; lanes C1–6, Δ_exsCD_; and lanes D1–6, Δ_exsCD_ additionally transformed with an ExsC expression plasmid (p_exsC_). Control lanes: Ec, soluble cell-associated fraction from E. coli overexpressing ExsEHis-6 (arrows); V, soluble cell-associated fraction from an Δ_exsD_ mutant carrying the plasmid control pJN105.

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