ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.

The opportunistic pathogen Pseudomonas aeruginosa ranks among the leading causes of nosocomial infections.  The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins (effectors) 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 ExsA-ExsC-ExsD-ExsE signaling cascade ties host cell contact to the up-regulation of T3SS gene expression.  Prior to T3SS induction, the antiactivator 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. ExsA is an AraC/XylS-type transcriptional regulator and belongs to a subfamily of activators that regulate the T3SS in a variety of Gram-negative pathogens.  These regulators are characteristically difficult to purify due to the low solubility of their C-terminal DNA binding domains.  A new method for purifying ExsA was developed and produced ExsA with improved solubility.  The interaction of ExsA and its PexsD promoter was examined using fluorescence anisotropy.  An in vitro transcription assay was developed and it was determined that ExsA is sufficient to activate T3SS transcription. Next, the ExsD--ExsA inhibitory mechanism was examined.  It was demonstrated for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in  vitro without the aid of any other cellular factors.  More significantly and contrary to previously published results, it was discovered that independently folded ExsD and ExsA are capable of interacting, but only at 37 degrees C and not at 30 degrees C.  Guided by the crystal structure of ExsD, a monomeric variant of the protein was designed to demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30 degrees C. To further elucidate the ExsD-ExsA inhibitory mechanism, the ExsD-ExsA interface was examined.  ExsD variants were generated and used to determine which region of ExsD interacts with ExsA.  Interestingly, ExsD was also found to bind DNA, although it is unclear whether or not this plays a role in ExsA inhibition.  Fully understanding the mechanism by which ExsD inhibits ExsA may enable the development of drugs that target ExsA in order to shut down the T3SS, thereby eliminating P. aeruginosa infection. / Ph. D.

Pseudomonas aeruginosa is a gram-negative pathogen that causes opportunistic infections in immunocompromised individuals. Whereas clinical isolates from acute infections are characterized by host cell cytotoxicity and motility, isolates from chronic infections are characterized by biofilm formation and persistence. The type III secretion system (T3SS) causes cytotoxicity by injecting effectors into host cells. T3SS gene expression is activated by ExsA, an AraC family transcriptional regulator. Transcription of exsA is controlled by two promoters, PexsC and PexsA, which are regulated by ExsA and the cAMP-Vfr system, respectively. Additional global regulatory systems also influence T3SS including the second messenger signaling molecule c-di-GMP and the RsmAYZ regulatory system. c-di-GMP signaling increases biofilm production and decreases acute virulence factor expression. A previous study found that c-di-GMP alters cAMP levels and affect cAMP-Vfr signaling. Other studies found that c-di-GMP signaling alters expression of the small non-coding regulatory RNAs, rsmY and rsmZ. The RsmAYZ post-transcriptional regulatory system regulates ExsA translation. We hypothesize that c-di-GMP regulates T3SS expression by altering exsA transcription through the cAMP-Vfr dependent PexsA promoter. Overexpression of YfiN, a c-di-GMP synthase, decreases T3SS reporter activity in PA103 and requires a functional GGDEF active site for full inhibition. Inhibition by YfiN does not require rsmYZ. YfiN expression decreases cAMP-Vfr signaling and coordinately inhibits PexsA-lacZ reporter activity. Consistent with the proposed model, YfiN expression in a vfr mutant does not further decrease T3SS reporter activity. These data indicate that the YfiN alters T3SS expression through transcriptional control of the cAMP-Vfr dependent PexsA promoter.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes acute nosocomial infections as well as chronic infections in cystic fibrosis (CF) patients. P. aeruginosa utilizes a type III secretion system (T3SS) during acute infections to promote host cell cytotoxicity and inhibit phagocytosis. Regulation of T3SS expression can be classified into two distinct categories: intrinsic and extrinsic. T3SS intrinsic regulation involves the well-characterized ExsECDA cascade that controls T3SS gene transcription. Extrinsic regulation involves global regulatory systems that affect T3SS expression. Despite general knowledge of global regulation of T3SS expression, few specific mechanisms have been elucidated in detail. The overall goal of my thesis work was to provide clarity to global regulatory mechanisms controlling T3SS expression. One well-documented observation is that P. aeruginosa isolates from CF patients commonly have reduced T3SS expression. In chapter II, I describe how the MucA/AlgU/AlgZR system, commonly activated in CF isolates through mutation of the mucA gene, inhibits T3SS gene expression. My experiments demonstrate that the AlgZR two-component system inhibits ExsA expression through two separate global regulatory systems. First, as previously described, AlgZR inhibits ExsA expression by reducing activity of the cAMP/Vfr signaling pathway. Vfr, a homolog of Escherichia coli Crp, regulates T3SS gene expression through an unknown mechanism. Second, AlgZR alters the activity of the RsmAYZ system to specifically reduce ExsA expression. The RNA-binding protein RsmA, a homolog of E. coli CsrA, activates ExsA expression at a post-transcriptional level. Previous studies in our laboratory identified several transposon insertion mutants that appeared to be novel extrinsic regulators of T3SS gene expression. One of those candidates, named DeaD, is a putative ATP-dependent RNA helicase. My experiments in chapter III reveal that DeaD regulates T3SS expression by directly stimulating exsA translation. Mutants lacking deaD have reduced exsA translational reporter activity and ExsA expression in trans fails to complement a deaD exsA double mutant for T3SS gene expression. I demonstrate that purified DeaD stimulates ExsA expression in a coupled in vitro transcription/translation assay, confirming our in vivo findings. In chapter II, I observed that RsmA activates the transcription of RsmY and RsmZ, two small non-coding RNAs that act to sequester RsmA from target mRNAs. My experiments in chapter IV begin to dissect the RsmA-activation mechanism of RsmY/Z expression. I show that RsmA activation requires the previously described Gac/Lad/Ret system that controls RsmY/Z expression. RsmA, however, does not alter Gac/Lad/Ret gene transcription or translation. Interestingly, an RsmA variant deficient in RNA-binding, RsmA R44A, was able to complement an rsmA mutant for RsmY/Z expression. I hypothesized that RsmA interacts with an unknown protein to activate RsmY/Z expression and identified several potential interaction partners using co-purification assays. Together, my combined experiments elucidate novel global regulatory pathways controlling T3SS gene expression during acute and chronic P. aeruginosa infections, and provide a foundation towards the goal of developing future treatment options.

Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. ExsA is comprised of an amino terminal domain that is involved in self-association and regulatory functions, and a carboxy-terminal domain that contains two helix-turn helix (HTH) DNA-binding motifs which contact promoter DNA. Previous work from our lab determined the function of the two independent ExsA domains and found that each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two HTH DNA-binding motifs of an ExsA monomer. Nevertheless, the details of how ExsA recognizes and binds to ExsA-dependent promoters were still unknown. In chapter II I show that the two ExsA monomers bind to promoter regions in a head-to-tail orientation and identify residues in the first HTH of ExsA that contact the GnC sequence. Likewise, residues located in the second HTH motif, which contribute to the recognition of the TGnnA sequence, were also identified. While the GnC and TGnnA sequences are important for binding to site 1, the promoter-distal binding sites (site 2) lack obvious similarity among themselves or with binding site 1. Site 2 in the PexsC promoter region contains a GnC sequence that is functionally equivalent to the GnC in site 1 and recognized by the first HTH motif of an ExsA monomer and the second HTH interacts with an adenine residue in binding site 2. A comparison of hybrid promoters composed of binding site 2 from one promoter fused to binding site 1 derived from another promoter indicates that ExsA-binding affinity, promoter strength, and the degree of promoter bending are properties that are largely determined by binding site 2. Through the course of the ExsA studies I observed that the amino acids that comprise the HTH motifs of ExsA are nearly identical to those in LcrF/VirF, the activators of T3SS gene expression in the pathogenic yersiniae. In chapter III I tested the hypothesis that ExsA/LcrF/VirF recognize a common nucleotide sequence. Here I report that Yersinia pestis LcrF binds to and activates transcription of ExsA-dependent promoters in P. aeruginosa, and that plasmid expressed ExsA complements a Y. pestis lcrF mutant for T3SS gene expression. Mutations that disrupt the ExsA consensus-binding sites in both P. aeruginosa and Y. pestis T3SS promoters prevent activation by ExsA and LcrF. All of the data combined demonstrate that ExsA and LcrF recognize a common nucleotide sequence. Nevertheless, the DNA binding properties of ExsA and LcrF are distinct. Whereas two ExsA monomers are sequentially recruited to the promoter region, LcrF binds to promoter DNA as a preformed dimer and has a higher capacity to bend DNA. An LcrF mutant defective for dimerization bound promoter DNA with properties similar to ExsA. Finally, I demonstrate that the activators of T3SS gene expression from Photorhabdus luminescens, Aeromonas hydrophila, and Vibrio parahaemolyticus are also sensitive to mutations that disrupt the ExsA-consensus binding site. Taken together, this work shows that ExsA binding and activation at T3SS gene promoters serves as a model system by which the DNA binding properties of other AraC family transcriptional activators can be predicted.

The Type III secretion system (T3SS) of Pseudomonas aeruginosa uses a needle-like protein apparatus to detect eukaryotic host cells and translocate effectors directly into the host cell. The effectors are also known as cytotoxins, which cause disruption of a series of signaling events in the host cell, facilitating the infection by P. aeruginosa. As the T3SS is antigenic and the expression of T3SS is energy-consuming, it is highly regulated where several regulatory proteins interact with each other and control the expression of T3SS genes. Among these proteins, ExsA, the master regulator of T3SS in P. aeruginosa, is of great importance as it is a transcriptional activator that activates the expression of all T3SS genes. Also, as ExsA belongs to the AraC protein family which only exists in bacteria and fungi, it makes an excellent potential target for drugs against P. aeruginosa related infections. With a combination of molecular biology tools and structural biology methods, we solved the N-terminal domain structure of the ExsA protein in P. aeruginosa. The model of the ExsA N-terminal domain has enriched our knowledge about ExsA dimerization and can serve as the base for mapping the interaction interfaces on ExsA and ExsD. Further, we have found two homologues of ExsA by structural alignment, which share a lot of similarities and have conserved amino acid residues that are important for ligand binding. The fact that both of these two proteins are regulated by small ligands rather than proteins also raises the possibility that ExsA may have a second regulatory mechanism under which ExsA is regulated by a small ligand, which so far has not been observed or reported by researchers. In order to map the binding site of ExsA on its anti-activator ExsD, we removed the coiled-coil region (amino acid residue 138-202, the potential binding site) of ExsD, based on the  structure of ExsD. We surprisingly found that the ExsD variant without the coiled-coil region readily inhibits ExsA-dependent in vitro transcription. This result rules out other possibilities and makes us focus on the N-terminus and adjacent regions of ExsD for the interface with ExsA. Moreover, in order to gain a comprehensive understanding of the dynamics of the regulation of T3SS in P. aeruginosa, we have begun to build a mathematical model of the T3SS regulatory pathways. We are measuring the cellular concentrations of T3SS regulatory proteins with quantitative molecular biology methods such as quantitative western blot, quantitative PCR and quantitative mass spectrometry. We have determined the cellular level of ExsA and ExsD proteins under different physiological conditions, and found that some factors such as temperature have a significant impact on the levels of ExsA and ExsD. This study has thus unveiled some unknown features of the T3SS of P. aeruginosa and its related infections. / Master of Science

L'expression des gènes codant pour le Système de Sécrétion de Type III (SST3), facteur de virulence majeur de Pseudomonas aeruginosa, est activée par ExsA. Ce facteur de transcription appartient à la famille des régulateurs de type AraC/XylS caractérisés par un domaine de fixation à l'ADN comportant deux motifs hélice-tour-hélice. L'activité de ces protéines est généralement régulée par la fixation d'un ligand sur un domaine supplémentaire non conservé. Ce ligand peut être de nature protéique dans le cas de certains régulateurs contrôlant la synthèse de SST3. Ainsi, l'activité transcriptionnelle de ExsA est inhibée par l'anti-activateur ExsD. L'étude de la fonctionnalité de ExsA et de ses domaines par des approches in vitro et in vivo a révélé que le domaine C-terminal de ExsA est bien le domaine de fixation à l'ADN et que deux monomères se fixent sur le promoteur de l'opéron des gènes de régulation du SST3 (pC). Ce domaine isolé possède une affinité pour pC et une activité transcriptionelle inférieure à celle de ExsA. En effet, la fixation efficace de ExsA sur le promoteur pC requiert sa dimérisation à travers son domaine N-terminal. La dernière hélice  de ce domaine N-ter semble jouer un rôle majeur dans la dimérisation de ExsA. Le deuxième objectif de ma thèse était de comprendre l'interaction entre ExsA et ExsD et d'identifier le mécanisme grâce auquel l'inhibiteur empêche ExsA d'activer la transcription des gènes du SST3. Après co-production des deux protéines, le complexe ExsA/ExsD a été purifié puis caractérisé. ExsA et ExsD forment un complexe hétérodimérique au sein duquel l'inhibiteur empêche le facteur de transcription de se fixer à l'ADN.