Self-Association Is Required for Occupation of Adjacent Binding Sites in Pseudomonas aeruginosa Type III Secretion System Promoters

Abstract

ExsA is a member of the AraC/XylS family of transcriptional regulators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). All P. aeruginosa T3SS promoters contain two adjacent binding sites for monomeric ExsA. The amino-terminal domain of ExsA (NTD) is thought to mediate interactions between the ExsA monomers bound to each site. Threading the NTD onto the AraC backbone revealed an ␣-helix that likely serves as the primary determinant for dimerization. In this study, we performed alanine scanning mutagenesis of the ExsA ␣-helix (residues 136 to 152) to identify determinants required for self-association. Residues L137, C139, L140, K141, and L148 exhibited self-association defects and were required for maximal activation by ExsA. Disruption of self-association resulted in decreased binding to T3SS promoters, particularly loss of binding by the second ExsA monomer. Removing the NTD or increasing the space between the ExsA-binding sites restored the ability of the second ExsA monomer to bind the P exsC promoter. This finding indicated that, in the absence of self-association, the NTD prevents binding by a second monomer. Similar findings were seen with the P exoT promoter; however, binding of the second ExsA monomer in the absence of self-association also required the presence of a high-affinity site 2. Based on these data, ExsA self-association is necessary to overcome inhibition by the NTD and to compensate for low-affinity binding sites, thereby allowing for full occupation and activation of ExsA-dependent promoters. Therefore, ExsA self-association is indispensable and provides an attractive target for antivirulence therapies.

Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N -Hydroxybenzimidazoles

“…2A lane 2), ExsA CTD more readily forms shift product 1 (i.e., binding of an ExsA monomer to a single site), owing to a lack of self-association between the ExsA monomers (Fig. 2D, lane 2) (39). Treatment of ExsA CTD with each of the N-hydroxybenzimidazoles resulted in decreased formation of shift products 1 and 2 (Fig.…”

Fitting Pieces into the Puzzle of Pseudomonas aeruginosa Type III Secretion System Gene Expression

“…1) (35,36,(39)(40)(41). Liberated ExsA then binds to target promoters and recruits RNA polymerase to activate the T3SS regulon (28,42,43). Partner switching is likely driven by affinity differences in the proteinprotein interactions (ExsE-ExsC Ͼ ExsD-ExsE Ͼ ExsA-ExsD, in decreasing order of affinity).…”

Characterization of ExsA self-association and DNA-binding as therapeutic targets to prevent expression of the Pseudomonas aeruginosa type III secretion system

“…This study monomeric ExsA is still able to occupy single promoter binding sites, ruling out the possibility that inhibiting self-association is the sole activity of ExsD (Fig. 6A) (276).…”

“…17A lane 2), ExsACTD more readily forms shift product 1 (i.e., binding of an ExsA monomer), owing to a lack of self-association between the ExsA monomers(Fig. 17D, lane 2)(276). In each case, treatment of ExsACTD with the Nhydroxybenzimidazoles resulted in decreased formation of shift products 1 and 2 (Fig.…”

“…Since bothExsA-ExsD complex formation and ExsA self-association are dependent upon the NTD, Ihypothesized that ExsD interacts with monomeric ExsA at the dimeric interface to sterically hinder self-association, and predicted that some of the alanine substitutions previously constructed in the self-association helix would interfere with ExsA-ExsD complex formation(276). Mutations that weaken complex formation, therefore, should result in increased reporter activity.…”