Nordstrom, A. | bioRxiv Science

bioRxiv2024

Extracellular ATP is a signalling molecule in bacteria

In animals and plants extracellular ATP (eATP) functions as signal and regulates the immune response. During inflammation intestinal bacteria are exposed to elevated eATP originating from the mucosa. However, whether bacteria respond to eATP is unclear. Here we show that non-pathogenic Escherichia coli responds to eATP by modifying its transcriptional and metabolic landscapes. A genome-scale promoter library showed that the response is time-, concentration-, and medium-dependent and ATP-specific. The second messenger cAMP and genes related to metabolism, quorum sensing, and envelope stress were regulated downstream of eATP. Metabolomics confirmed that eATP triggers enrichment of compounds with bioactive properties on the host or bacteria. Combined genome-scale modelling revealed global metabolic and biomass building blocks modifications. Consequently, eATP altered the sensitivity to antibiotics and antimicrobial peptides. Finally, in pathogens eATP controlled virulence factor expression. Our results indicate that eATP is an environmental cue in prokaryotes which broadly regulates physiology, antimicrobial resistance, and virulence.

microbiology↗

bioRxiv2018

14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface

Pseudomonas are a common cause of hospital acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, a hydrophobic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3{beta}:ExoS and -ExoT complexes presented here reveal an extensive novel binding interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the hydrophobic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the hydrophobic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the hydrophobic C-terminal segment.\n\nShort summaryCrystal structures of Pseudomonas exotoxins-S and -T identify a novel hydrophobic interface with 14-3-3 proteins, and we show that 14-3-3 activates these toxins independent of their phosphopeptide groove binding C-termini, by preventing their aggregation.

biochemistry↗