Genetic Analysis of Allosteric Signaling in RhaR from Escherichia coli and Characterization of the VirF Protein from Shigella flexneri

Abstract

The RhaR and VirF proteins are both members of the large AraC family of bacterial transcription regulators. RhaR activates expression of the Escherichia coli rhaSR operon in response to the effector L-rhamnose while VirF is the master regulator of expression of the Shigella flexneri type three secretion system (T3SS) and activates transcription in response to temperature and pH. Both proteins consist of two domains: an N-terminal domain (NTD) and a conserved DNA binding domain (DBD) responsible for binding to DNA and contacting RNA polymerase (RNAP) to activate transcription. The RhaR NTD is responsible for protein dimerization and binding the effector L-rhamnose and is required for maximal transcription activation. The VirF NTD is currently uncharacterized, but has been hypothesized to be involved in oligomerization (likely dimerization) of the protein. The principal goals of this study were to further define the mechanism of RhaR interdomain allosteric signaling and to characterize the general mechanisms of transcription activation by VirF. In the current study, we sought to further elucidate the mechanism of allosteric signaling in RhaR that mediates the response to L-rhamnose. My approach was to examine the role of residues predicted to make interdomain contacts between the RhaR N-terminal domain (NTD) and DNA binding domain (DBD). I generated mutations to examine the role of residues in two regions of the DBD: Allosteric site in subdomain 2 (AS2) and the C-termini of the two helix-turn-helix motifs (C-HTH1 and C-HTH2). At AS2, results indicated that one residue may be involved in inhibitory contacts that reduce the activity of RhaR minus rhamnose. Furthermore this residue likely interacts with a residue in the RhaR Arm to inhibit transcription activation minus rhamnose. This conclusion is supported by the isolation of a second-site suppressor mutation in AS2 that restored activity of an Arm variant to wild-type levels. We propose that in the absence of L-rhamnose contacts between the RhaR Arm (in the NTD) and AS2 (in the DBD) regions minus rhamnose that constrain the RhaR conformation such that it is unable to efficiently activate transcription. We also sought to better characterize the Shigella master virulence regulator, VirF. I first investigated the ability of the isolated VirF DBD to activate transcription. The isolated VirF DBD did not activate transcription above background levels, indicating the DBD is not sufficient to activate transcription in the absence of the NTD. We then investigated the role of the VirF NTD. Structural modeling of the VirF protein showed that the NTD of VirF may have structural similarity to the NTDs of the AraC and ToxT proteins, both of which are responsible for dimerization and effector binding. I subsequently screened for potential effectors of VirF and further investigated the oligomeric state of VirF. Preliminary results indicated that VirF likely forms dimers in solution in addition to binding to DNA as a dimer. Furthermore, the effector screen identified bicarbonate as a potential repressor of VirF activity, although more studies are necessary to confirm the role of bicarbonate in VirF activation. Nonetheless, I propose a model where bicarbonate may serve as a spatial regulator of expression of the Shigella T3SS, aiding in navigation of the organism to the large intestine where the organism invades the epithelial cells, establishing infection. The last goal of this study was to investigate inhibition of RhaR and VirF by the small-molecule inhibitor SE-1 in vitro. SE-1 inhibits transcription activation in vivo and DNA binding in vitro of a closely related AraC family regulator, RhaS. I performed electrophoretic mobility shift assays in the presence or absence of SE-1 to determine the ability of the inhibitor to block DNA binding by either RhaR or VirF. I found that SE-1 was able to inhibit in vitro DNA binding by RhaR in a dose-dependent manner. Preliminary studies indicated that SE-1 also inhibited VirF in a dose-dependent manner. From our collective results, we propose that SE-1 blocks transcription activation of RhaS, RhaR and VirF by binding to the conserved DBD and blocking DNA binding. Binding of SE-1 to the conserved DBD that defines the AraC family of activators supports the hypothesis that SE-1 may inhibit other AraC family regulators, providing potential for development as a novel broad-spectrum anti-infective.