Transcriptional control of virulence in Francisella tularensis

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2022

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One of the most infectious bacteria in the world, the Gram-negative bacterium Francisella tularensis is the etiological agent of tularemia in humans. Francisella virulence stems from a gene cluster known as the Francisella pathogenicity island that encodes a type VI secretion system and enables it to escape from macrophages during an infection. Expression of the FPI is linked to the stringent response and is tightly controlled. Three transcription factors, macrophage growth locus protein A (MglA), stringent starvation protein A (SspA), and the pathogenicity island gene regulator (PigR), form a complex with RNA polymerase (RNAP) to activate transcription of the FPI. The alarmone of the stringent response, ppGpp, mediates the formation of the regulatory complex. MglA, which is unique to Francisella, forms a heterodimer with the conserved factor SspA, which functions as a homodimer in other Gram-negatives. Together, the MglA-SspA heterodimer associates with the σ70-containing RNAP using its closed face and binds ppGpp directly on the opposite face. With ppGpp bound, PigR can bind MglA-SspA with its C-terminal end while also making contacts to an upstream promoter DNA site known as the PigR response element (PRE) likely using its putative winged helix-turn-helix motif. Once the full complex is assembled, FPI transcription is activated through mechanisms that are not well understood. It is unclear how MglA-SspA, and PigR associate with RNAP and how these factors drive transcription at virulence promoters and, thus, enable Francisella to cause disease. To provide insight into the mechanisms of virulence activation by this unusual set of regulators, we employed single-particle cryo-EM, X-ray crystallography, and cellular analyses. First, we determined a cryo-EM structure of F. tularensis RNAPσ70 in complex with MglA-SspA and a virulence promoter which revealed the MglA-SspA binding mechanism. MglA-SspA does not contact the DNA, but instead interacts with two regions of the σ factor and makes an extensive hydrophobic interface with the RNAP core subunit β’. This effectively tethers the σ factor to the core enzyme to stabilize the RNAP holoenzyme and facilitate DNA binding. This structure adopted an open-complex; however, when we determined the structure with the same virulence promoter in the absence of MglA-SspA, the complex was unable to stably bind the DNA suggesting that MglA-SspA may itself regulate transcription at certain promoters by facilitating promoter binding and possibly open complex formation. We followed up on this with RNA sequencing studies that demonstrated MglA-SspA regulates many virulence and virulence-enhancing non-FPI genes independently of PigR. We next determined a cryo-EM reconstruction of E. coli RNAP in complex with an E. coli SspA homodimer and DNA which showed conservation of the σ-tethering mechanism. While we showed MglA-SspA alone can regulate a subset of virulence and virulence-enhancing genes, the activation of FPI genes requires PigR. We determined a crystal structure of (MglA-SspA)-ppGpp with a PigR C-terminal tail peptide. PigR makes direct contact to ppGpp through electrostatic interactions, which explains why ppGpp is needed for specific binding of PigR. Lastly, a cryo-EM structure of the full virulence complex consisting of Ftu RNAPσ70-(MglA-SspA)-ppGpp-PigR with an FPI promoter revealed an unexpected activation mechanism. This reconstruction revealed that PigR enhances transcription by recruiting the C-terminal domains of the RNAP core α subunits to previously unrecognized DNA UP elements that flank the PRE on both sides. All together, these findings have significantly advanced our understanding of virulence activation in F. tularensis and shed light on a general mechanism of transcription regulation by the SspA protein family.

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Travis, Brady Andrew (2022). Transcriptional control of virulence in Francisella tularensis. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25214.

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