Molecular elucidation of the Francisella tularensis virulence mechanism

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Francisella tularensis (Ft), the causative agent of tularemia, is one of the most infectious bacterial pathogens known. Due to its high infectivity and ease of aerosolization, it has been classified as Category A bioweapon by the US government. The morbidity and mortality of tularemia are substantial, and given its extreme infectivity, a significant outbreak of tularemia would readily overwhelm the capabilities of even the largest US medical centers. Ft virulence requires genes expressed from the chromosomally encoded Ft pathogenicity island (FPI). The expression of these genes are activated by a combination of Ft regulators: the stringent starvation protein A (SspA), the macrophage growth locus protein A (MglA) and the pathogenicity island gene regulator (PigR), which are expressed during Ft infection. MglA and PigR are unique to Ft whereas SspA proteins are found in multiple bacteria. The Ft SspA, however, is unusual as it does not homodimerize but rather heterodimerizes with MglA. PigR is a DNA-binding protein with a predicted winged-helix-turn-helix DNA-binding motif. Intriguingly, the “alarmone”, guanosine tetraphosphate (ppGpp), is also necessary for Ft virulence. Recently, we showed that this alarmone binds directly to the MglA-SspA complex. We further showed that ppGpp binding to MglA-SspA mediates high-affinity binding of PigR to this heterodimer. Strikingly, our data also revealed that MglA-SspA interacts constitutively with the Ft RNAPs70 holoenzyme suggesting it represents a virulence specialized RNAP. Given the extreme virulence and potential use of Ft as a bioweapon, there is an urgent need to decipher the molecular mechanisms driving its virulence. The overarching goal of this proposal is the molecular dissection of these mechanisms. Our central hypothesis is that Ft employs a conceptually novel form of pathogenesis requiring a virulence-specialized RNAP containing MglA-SspA. We shall test our central hypothesis and delineate the molecular mechanisms controlling the activation of Ft virulence genes through the completion of three Specific Aims: Specific Aim 1: To fully characterize MglA-SspA interaction with Ft RNAP. Structural, biochemical and cellular studies will dissect the mechanism of virulence regulation by MglA- SspA. Specific Aim 2: To carry out structure and function analyses of Ft RNAP(MglA-SspA)-ppGpp-PigR complexes. The mechanism behind PigR-mediated activation of the FPI will be analyzed structurally, biochemically and in vivo. Specific Aim 3: To determine a high resolution (MglA-SspA)-ppGpp-PigR crystal structure, identify inhibitors of ppGpp binding to MglA-SspA and obtain structures of MglA-SspA inhibitor complexes. The successful completion of these Aims will reveal new paradigms in transcription regulation and enable the rational design of novel anti Francisella-virulence therapeutics.