Identification of a Small Molecule That Modifies MglA/SspA Interaction and Impairs Intramacrophage Survival of <em>Francisella tularensis</em>

<div><p>The transcription factors MglA and SspA of <em>Francisella tularensis</em> form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the <em>Francisella</em> pathogenicity island (FPI) genes. These genes are essential for this pathogen’s virulence and survival within host cells. In this study, we used a small molecule screening to identify quinacrine as a thermal stabilizing compound for <em>F. tularensis</em> SCHU S4 MglA and SspA. A bacterial two-hybrid system was used to analyze the <em>in vivo</em> effect of quinacrine on the heterodimer complex. The results show that quinacrine affects the interaction between MglA and SspA, indicated by decreased β-galactosidase activity. Further <em>in vitro</em> analyses, using size exclusion chromatography, indicated that quinacrine does not disrupt the heterodimer formation, however, changes in the alpha helix content were confirmed by circular dichroism. Structure-guided site-directed mutagenesis experiments indicated that quinacrine makes contact with amino acid residues Y63 in MglA, and K97 in SspA, both located in the “cleft” of the interacting surfaces. In <em>F. tularensis</em> subsp. <em>novicida</em>, quinacrine decreased the transcription of the FPI genes, <em>iglA, iglD, pdpD</em> and <em>pdpA</em>. As a consequence, the intramacrophage survival capabilities of the bacteria were affected. These results support use of the MglA/SspA interacting surface, and quinacrine’s chemical scaffold, for the design of high affinity molecules that will function as therapeutics for the treatment of Tularemia.</p>