Date of Degree
PhD (Doctor of Philosophy)
Timothy L. Yahr
Regulation of the Pseudomonas aeruginosa type III secretion system (T3SS) is controlled by the transcriptional activator ExsA, a member of the AraC family of regulators. Members of this family are characterized by a conserved DNA-binding domain, which contains two helix-turn-helix DNA-binding motifs and is generally located in the carboxy-terminal domain (CTD). Previous work from our lab has characterized the interaction between promoter DNA and the ExsA CTD. Two monomers of ExsA bind promoter DNA at binding sites 1 and 2 (centered at -41 and -65 relative to the transcriptional start site, respectively) to recruit RNA polymerase and activate transcription of all T3SS promoters. This interaction is required for T3SS gene expression, making it an attractive target for inhibitors designed to disrupt T3SS activity. In this study, I have characterized a group of N-hydroxybenzimidazole compounds that disrupt the ExsA-DNA interaction, leading to decreased gene expression and T3SS-mediated cytotoxicity. Furthermore, N-hydroxybenzimidazoles interact with the ExsA DNA-binding domain, and due to the conserved nature of this domain, these compounds have broad-spectrum activity against ExsA homologs.
The amino-terminal domain (NTD) of AraC family proteins is poorly conserved at the primary amino acid sequence level; however, oligomerization and/or ligand binding is commonly mediated by the NTD of AraC family proteins. The ExsA NTD is required for both self-association and interaction with the anti-activator protein ExsD. In addition to DNA-binding by the ExsA CTD, I have shown that ExsA self-association is required for maximal activation of T3SS promoters, providing another target for ExsA inhibitors. In the current model for ExsA interactions with promoter DNA, ordered occupation of binding site 1 followed by occupation of binding site 2 is facilitated by an interaction between ExsA monomers. In this study, I identified an α-helix required for ExsA self-association and showed that ExsA self-association serves two distinct roles to promote occupation of binding site 2 by a second ExsA monomer: (1) self-association relieves NTD-mediated inhibition of site 2 occupation, and (2) self-association facilitates occupation of low-affinity binding sites.
Lastly, the interaction between ExsD and ExsA prevents both ExsA self-association and DNA-binding, resulting in loss of activation at T3SS promoters. An understanding of the molecular basis for ExsA inhibition by ExsD is unknown but could provide valuable insight for the development of ExsA inhibitors. I performed site-directed and random mutagenesis to identify ExsA residues involved in the ExsA-ExsD interaction. Residues required for this interaction were identified within the ExsA self-association helix and elsewhere in the NTD; therefore, ExsD appears to prevent ExsA self-association by contacting multiple regions of ExsA, including the self-association helix. As a whole, these studies were performed to further our understanding of the protein-protein and protein-DNA interactions that are integral to the regulatory activity of ExsA and to provide direction for the development of therapeutic strategies that prevent expression of the P. aeruginosa T3SS.
Pseudomonas aeruginosa is a common cause of bacterial infections acquired by immunocompromised individuals in healthcare facilities. A major strategy employed by P. aeruginosa to cause infections is the injection of toxins directly into host cells through a needle-like structure on the bacterial cell surface. This structure is called a type III secretion system (T3SS), and its expression is controlled by the regulatory protein ExsA. Without ExsA, the T3SS is not expressed, making ExsA an attractive target for therapeutic strategies to prevent P. aeruginosa infections.
The T3SS is composed of many proteins, each the product of a gene whose transcription depends on ExsA. Two molecules of ExsA bind to a region of DNA next to T3SS genes and recruit RNA polymerase, activating transcription. I have shown that an interaction between the two molecules of ExsA (called self-association) is required for maximal binding and activation of T3SS genes. This interaction is mediated by a specific region of ExsA, which may serve as a target for small-molecule inhibitors of ExsA activity.
An alternative way to inhibit ExsA activity is to directly prevent DNA-binding. A group of compounds called N-hydroxybenzimidazoles have been identified as inhibitors of ExsA and other closely related proteins. I showed that these compounds prevent T3SSmediated effects by inhibiting DNA-binding and transcriptional activation by ExsA. Lastly, another regulatory protein, ExsD, can prevent DNA-binding and self-association by ExsA. I have started to characterize the interaction between ExsA and ExsD in order to use this interaction as a model for future development of ExsA inhibitors.
xii, 177 pages
Includes bibliographical references (pages 155-177).
Copyright 2015 Anne Elizabeth Marsden