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.

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.