Project description:The bacterium, Francisella tularensis (Ft), is one of the most infectious agents known and classified as a category A bioweapon. Ft virulence is controlled by a unique set of transcription regulators, the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence. MglA-SspA is expressed during infection and constitutively associates with the σ70 associated RNAP holoenzyme (RNAPσ70), indicating that RNAPσ70-(MglA-SspA) is a virulence specific polymerase. How virulence activation is mediated by these components, however, is unknown. Here we report cryo-EM structures of FtRNAPσ70, FtRNAPσ70-(MglA-SspA) and RNAPσ70-(MglA-SspA)-ppGpp-PigR complexes with promoter DNA. FtRNAPσ70-DNA and FtRNAPσ70-(MglA-SspA)-DNA structures and RT-PCR analyses show MglA-SspA stabilizes σ70 binding to DNA to regulate FPI-independent, virulence-enhancing genes. Strikingly, an Escherichia coli RNAPσ70 complex with EcSspA suggests this is a general mechanism for SspA-like regulation of bacterial RNAPσ70. Finally, our FtRNAP-σ70-(MglA-SspA)-ppGpp-PigR-DNA structure reveals that ppGpp binds to MglA-SspA to tether the DNA-binding activator, PigR, to FPI promoters. PigR in turn recruits FtRNAP CTDs to two DNA upstream (UP) elements, generating stable FPI transcription complexes. Thus, these studies unveil a novel paradigm for pathogenesis in Ft involving a virulence-specific RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
2020-11-05 | GSE150932 | GEO
Project description:CRISPRi screen and whole genome sequencing
Project description:The IncX3 plasmid is a primary vector for disseminating carbapenem resistance globally, yet the chromosomal regulatory mechanisms governing its fitness cost remain poorly understood. Our previous work identified the stringent starvation protein SspA as a modulator of IncX3 plasmid fitness in Escherichia coli via flagellar biosynthesis. Here, we elucidate the global regulatory role of SspA and verify whether its effect is mediated through the master regulator H-NS
Project description:Treatment of mouse skin with the staphylococcal protease SspA and house dust mite extract (HDM) results in dermatitis. Using a 10x Genomics scRNA-seq platform, we analyzed T cells isolated from inflamed skin after treatment with epicutaneous SspA and HDM.
Project description:Treatment of mouse skin with the staphylococcal protease SspA and house dust mite extract (HDM) results in dermatitis. Using a 10x Genomics Xenium In Situ platform, we analyzed spatial features in mock-treated skin and inflamed skin treated with epicutaneous SspA and HDM.