Structural evidence suggests that antiactivator ExsD from <i>Pseudomonas aeruginosa</i> is a DNA binding protein

Bernhards, Robert C.; Xing, Jing; Vogelaar, Nancy J.; Robinson, Howard; Schubot, Florian D.

doi:10.1002/pro.48

Cited by 17 publications

(24 citation statements)

References 53 publications

(70 reference statements)

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“…With the use of a calibration curve obtained by analyzing a set of standard proteins, the retention time of the variant protein gave an apparent molecular mass of 42 kDa. Although this is somewhat larger than the actual 32‐kDa mass of an ExsD monomer, the discrepancy is readily explained by the distinctively nonglobular shape of the molecule . In contrast, wild‐type ExsD (Fig.…”

Section: Resultsmentioning

confidence: 83%

“…A striking feature of ExsD is the apparent plasticity of its oligomeric state, depending on the interacting partner. ExsD forms a 2 : 2 complex with ExsC and a 1 : 1 complex with ExsA, whereas analytical ultracentrifugation studies and the ExsD crystal structure suggest that ExsD self‐associates to form a trimer in the absence of the other two proteins . Our recent work suggests that the ExsC–ExsD complex actually consists of two ExsD monomers bound to an obligate ExsC dimer, rather than a dimer of dimers .…”

Section: Resultsmentioning

confidence: 86%

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Self‐trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro

et al. 2013

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The opportunistic pathogen Pseudomonas aeruginosa ranks among leading causes of nosocomial infections. The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins into host cells to suppress the host's innate immune response. Expression of all T3SS-related genes is strictly dependent upon the transcription factor ExsA. Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest. The presented work focuses on the ExsA-ExsC-ExsD-ExsE signaling cascade that ties host cell contact to the up-regulation of T3SS gene expression. Prior to T3SS induction, the anti-activator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC resulting in the release of ExsA and an up-regulation of the T3SS. Previous studies have shown that the ExsD-ExsA interactions are not freely reversible. Because independently folded ExsD and ExsA were not found to interact, it has been hypothesized that folding intermediates of the two proteins form the complex. Here we demonstrate for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in vitro and that no other cellular factors are required. More significantly, we show that independently folded ExsD and ExsA are capable of interacting, but only at 37°C and not at 30°C. Guided by the crystal structure of ExsD, we designed a monomeric variant of the protein and demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30°C. We propose that this unique mechanism plays an important role in T3SS regulation.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 86%

Self‐trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro

et al. 2013

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“…The fact that ExsD is unable to bind ExsA unless both proteins are simultaneously expressed would suggest that de novo synthesis of ExsD is required for inhibition of T3SS gene expression. ExsD appears to have three fates when synthesized: ExsA binding, ExsC binding, or self-association into a trimer (1,37). One possibility to account for the data is that ExsD or ExsA preferentially binds to a folding intermediate of the cognate partner to form the 1:1 stoichiometric complex.…”

Section: Discussionmentioning

confidence: 99%

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

2010

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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.

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“…Curiously, the ExsD crystal structure revealed that the amino-terminus of ExsD has structural similarity to the DNA-binding domain of the KorB transcriptional repressor (Bernhards et al, 2009). The predicted DNA-binding interface, however, is buried within the trimeric structure suggesting that dissociation of the trimer might be required for ExsD to exhibit DNA-binding activity.…”

Section: Intrinsic Regulation Of T3ss Gene Expressionmentioning

confidence: 99%

Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa

Díaz¹

2011

Front. Microbio.

119

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Pseudomonas aeruginosa is an opportunistic pathogen that is particularly problematic in the healthcare setting where it is a frequent cause of pneumonia, bloodstream, and urinary tract infections. An important determinant of P. aeruginosa virulence is a type III secretion system (T3SS). T3SS-dependent intoxication is a complex process that minimally requires binding of P. aeruginosa to host cells, injection of the cytotoxic effector proteins through the host cell plasma membrane, and induction of T3SS gene expression. The latter process, referred to as contact-dependent expression, involves a well-characterized regulatory cascade that activates T3SS gene expression in response to host cell contact. Although host cell contact is a primary activating signal for T3SS gene expression, the involvement of multiple membrane-bound regulatory systems indicates that additional environmental signals also play a role in controlling expression of the T3SS. These regulatory systems coordinate T3SS gene expression with many other cellular activities including motility, mucoidy, polysaccharide production, and biofilm formation. The signals to which the organism responds are poorly understood but many seem to be coupled to the metabolic state of the cell and integrated within a master circuit that assimilates informational signals from endogenous and exogenous sources. Herein we review progress toward unraveling this complex circuitry, provide analysis of the current knowledge gaps, and highlight potential areas for future studies. Complete understanding of the regulatory networks that control T3SS gene expression will maximize opportunities for the development of strategies to treat P. aeruginosa infections.

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Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein

Cited by 17 publications

References 53 publications

Self‐trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro

Self‐trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro

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

Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa

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