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

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.

SUBMITTER: Urbanowski ML

PROVIDER: S-EPMC1175016 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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

Urbanowski Mark L ML Lykken Guinevere L GL Yahr Timothy L TL

Proceedings of the National Academy of Sciences of the United States of America 20050628 28

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 ...[more]

PMID: 15985546

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Project description:UnlabelledThe Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in avfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (PexsA) located immediately upstream of exsA PexsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a PexsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and PexsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the PexsA promoter, which is internal to the previously characterized exsCEBA operon.ImportanceVfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA.

Project description:UnlabelledThe opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.ImportanceThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Project description:Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA.IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.

Dataset Information

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

Publications

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

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