Dissection of the molecular circuitry controlling virulence in <i>Francisella tularensis</i>

Abstract

<i>Francisella tularensis,</i> the etiological agent of tularemia, is one of the most infectious bacteria known. Because of its extreme pathogenicity, <i>F. tularensis</i> is classified as a category A bioweapon by the US government. <i>F. tularensis</i> virulence stems from genes encoded on the <i>Francisella</i> pathogenicity island (FPI). An unusual set of <i>Francisella</i> regulators-the heteromeric macrophage growth locus protein A (MglA)-stringent starvation protein A (SspA) complex and the DNA-binding protein pathogenicity island gene regulator (PigR)-activates FPI transcription and thus is essential for virulence. Intriguingly, the second messenger, guanosine-tetraphosphate (ppGpp), which is produced during infection, is also involved in coordinating <i>Francisella</i> virulence; however, its role has been unclear. Here we identify MglA-SspA as a novel ppGpp-binding complex and describe structures of apo- and ppGpp-bound MglA-SspA. We demonstrate that MglA-SspA, which binds RNA polymerase (RNAP), also interacts with the C-terminal domain of PigR, thus anchoring the (MglA-SspA)-RNAP complex to the FPI promoter. Furthermore, we show that MglA-SspA must be bound to ppGpp to mediate high-affinity interactions with PigR. Thus, these studies unveil a novel pathway different from those described previously for regulation of transcription by ppGpp. The data also indicate that <i>F. tularensis</i> pathogenesis is controlled by a highly interconnected molecular circuitry in which the virulence machinery directly senses infection via a small molecule stress signal.