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. 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 F. tularensis SCHU S4. Analysis of the Mgla/SspA interaction was carried out using a two-hybrid system. The Escherichia coli reporter strain contained a deletion on the ppK-ppX 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 F. tularensis, chromatin immune precipitation studies revealed that in the absence of polyP, binding of the MglA/SspA complex to the promoter region of the pdpD, iglA, fevR and ppK genes is decreased. Isothermal titration calorimetry (ITC) indicated that polyP binds directly to the MglA/SspA complex with high affinity (KD = 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.
Highlights
Upon entry into host cells, pathogenic bacteria must evade the attack launched by the immune system, which is directed to eliminate invaders via the oxidative burst
Charity et al (2009) [19] hypothesized that ppGpp could control expression of MglA/SspA regulated genes. This metabolite is synthesized by the activity of the bifunctional (p)ppGpp synthase/hydrolase, SpoT, and the RelA ppGpp synthase, in their experiments with a F. holarctica DrelA DspoT mutant, no effects were observed on the association of the MglA/SspA complex with RNA polymerase (RNAP)
Due to the pleiotropic effects associated with the inability to synthesize ppGpp [22], we hypothesized that the accumulation of an intracellular metabolite could be responsible for the increased interaction of Ft-SspA/Ft-MglA observed in the AW24 strain
Summary
Upon entry into host cells, pathogenic bacteria must evade the attack launched by the immune system, which is directed to eliminate invaders via the oxidative burst. The persistence of microorganisms during periods of unfavorable environmental conditions, including abrupt changes in pH and decreased nutrient availability, is made possible by a series of metabolic changes and the expression of virulence genes that promote survival. The modulation of such expression is predominantly mediated by global transcriptional regulators. Similar results have been reported in the intracellular pathogens Shigella flexneri and Salmonella enterica [10] In these microorganisms, RpoS positively regulates the expression of proteins required to alleviate oxidative stress during host invasion, such as superoxide dismutase, peroxidases, and catalases [11,12]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
