MglA/SspA Complex Interactions Are Modulated by Inorganic Polyphosphate

<div><p>The transcription factors MglA and SspA of <i>Francisella tularensis</i> form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the <i>Francisella</i> pathogenicity island (FPI) genes. These genes are essential for this pathogen’s virulence and survival within host cells. Our goal was to determine if an intracellular metabolite modulate these protein/protein interactions. In this study, we identified inorganic polyphosphate (polyP) as a signal molecule that promotes the interaction of MglA and SspA from <i>F. tularensis</i> SCHU S4. Analysis of the Mgla/SspA interaction was carried out using a two-hybrid system. The <i>Escherichia coli</i> reporter strain contained a deletion on the <i>ppK-ppX</i> operon, inhibiting polyP synthesis. The interaction between MglA and SspA was significantly impaired, as was the interaction between the MglA/SspA complex and the regulatory protein, FevR, indicating the stabilizing effect of polyP. In <i>F. tularensis</i>, chromatin immune precipitation studies revealed that in the absence of polyP, binding of the MglA/SspA complex to the promoter region of the <i>pdpD, iglA, fevR and ppK</i> genes is decreased. Isothermal titration calorimetry (ITC) indicated that polyP binds directly to the MglA/SspA complex with high affinity (K_D = 0.3 µM). These observations directly correlated with results obtained from calorimetric scans (DSC), where a strong shift in the mid-transition temperature (Tm) of the MglA/SspA complex was observed in the presence of polyP.</p></div

PolyP concentrations in different mutant strains of <i>F. novicida</i>.

<p>PolyP concentrations were determined in <i>F. novicida</i> carrying the empty pKK214 plasmid (black bars) or pSAB (grey bars). PolyP concentrations were determined as described in material and methods. Statistical analyses were performed to compare the polyP concentrations in the wild type strain carrying pKK214, to the mutant strains carrying either pKK214 or pSAB. *p<0.05.</p

Oligonucleotides used in this study.

a<p>Underlines indicate the restriction sites.</p>b<p>Bold indicates the priming site.</p>c<p>Bold and underline indicate the His-tag.</p

PolyP is required for expression of pathogenicity determinants regulated by the MglA/SspA complex.

<p>Chromatin immune precipitation assays were performed with <i>F. novicida</i> cells and its mutant derivatives carrying the pSAB. The enrichment factor was calculated as the ratio of amplified DNA (promoter regions of <i>pdpD, iglA, ppK</i> and <i>fevR</i> genes) in <i>F. novicida</i> wild type strain, <i>mglA</i>, <i>ppK</i> and <i>sspA</i> mutants carrying pSAB over the strain carrying the empty pKK214 plasmid. Statistical analyses were performed on the enrichment factor obtained for strains carrying the pSAB over the strain carrying the empty pKK214 plasmid. *p<0.05.</p

In the absence of polyP, the interaction between FevR, MglA and SspA is impaired.

<p>Assays were performed with cells of the <i>E. coli</i> reporter strains AW23 (Δ<i>sspA</i>, square), AW24 (Δ<i>sspA</i> Δ<i>relA</i> Δ<i>spoT</i>, circle) and AW26 (Δ<i>sspA</i> Δ<i>ppKppX</i>, triangle) carrying the pBR-<i>mglA</i>-ω+pACTR-<i>fevR</i>-Zif+pCL-<i>sspA</i>. The β-galactosidase activity (expressed in arbitrary units, AU) was determined as described in material and methods. The basal level enzyme activity was subtracted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076428#pone.0076428.s002" target="_blank">Fig. S2</a>).</p

Polyphosphate binds the Ft-MglA/Ft-SspA complex with high affinity.

<p>(A) Chromatograms of Ft-MglA/Ft-SspA after the first dialysis cycle (continuous line) or after extensive dialysis (dotted line). (B) Effect of polyphosphate on the thermal unfolding of the Ft-MglA/Ft-SspA complex showing a shift in the transition temperature in the presence of 100 µM of polyphosphate. (C) Isothermal titration calorimetric data for the binding of polyphosphate to the Ft-MglA/Ft-SspA complex. For size exclusion chromatography, 100 µl protein samples in 10 mM Tris (pH 8), 500 mM NaCl were injected onto a prepacked Superose 12 10/300 GL gel filtration column. The DSC experiments were performed in 10 mM phosphate (pH 7.9), 500 mM NaCl in the absence (solid line) or with 100 µM (dashed line) of polyphosphate. For ITC, measurement of heat changes (upper panel) and integrated peak areas (lower panel) of a series of 5 µl injections of 100 µM polyphosphate, into a 16.7 µM protein solution, prepared in 10 mM Tris (pH 8.0), 150 mM NaCl. Experiments were carried out at 18°C.</p