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

Abstract

The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella 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 F. tularensis SCHU S4 MglA and SspA. A bacterial two-hybrid system was used to analyze the in vivo 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 in vitro 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 F. tularensis subsp. novicida, quinacrine decreased the transcription of the FPI genes, iglA, iglD, pdpD and pdpA. 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.

MglA/SspA Complex Interactions Are Modulated by Inorganic Polyphosphate

“…In the F. novicida Δ sspA mutant, a 3-fold decrease in ppK expression was observed. In the absence of MglA or SspA, ppK expression levels were similar to other previously identified pathogenicity genes whose expression is dependent on the MglA/SspA complex [21]. Conversely, no changes in the ppx mRNA levels were observed in any of the strains tested.…”

“…We have recently shown that the interaction between MglA and SspA can be manipulated using small molecules that bind in the cleft region of the heterodimer [21]. The goal of this work was to identify an intracellular metabolite capable of modulating these protein-protein interactions.…”

“…The strains were constructed as follows. For the AW23 strain, an E. coli sspA knockout mutant (AW18) was constructed in strain FW102 [46] using the protocol described by Datsenko and Wanner, (2000) [47] and reported in Wrench et al (2013) [21]. For AW24, the AW18 strain was used to delete the relA gene to obtain AW19, which was then used to delete the spoT gene, resulting in the AW20 strain.…”

Dissection of the molecular circuitry controlling virulence in Francisella tularensis

“…Previous data have shown that the Francisella SspA protein is not stable when expressed alone and requires MglA to be soluble (Wrench et al 2013a;Cuthbert et al 2015). In contrast, MglA can be readily expressed as a soluble protein and can form homodimers at high concentrations, although it preferentially interacts with SspA to form the physiologically active MglA-SspA complex that mediates FPI regulation (Charity et al 2007;Cuthbert et al 2015).…”

“…In other bacteria, SspA proteins form homodimers, and several of these proteins have been implicated in virulence, including the SspA proteins from enterohemorrhagic Escherichia coli (EHEC) (Williams et al 1994;Hansen et al 2005;Hansen and Jin 2012), Neisseria gonorrhoeae (DeReuse and Taha 1997), Vibrio cholerae (Merrell et al 2002;Xu et al 2003), and Yersinia enterocolitica (Badger et al 2000). The F. tularensis SspA protein, which shares ∼30% sequence identify with other bacterial SspAs, is not homodimeric and instead forms a complex with MglA (Charity et al 2007;Wrench et al 2013a;Cuthbert et al 2015). Interestingly, recent data showed that MglA also harbors a GST-like fold, although this protein shows little sequence homology with SspA proteins ; the F. tularensis MglA and SspA share only 19% sequence identity.…”