Novel RelA-SpoT Homolog toxin-antitoxin systems that inhibit bacterial growth through production of toxic alarmone ppApp

Abstract: The stress alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively known as (p)ppGpp, are the molecular mediators of the bacterial stringent response — a bacterial stress response implicated in virulence, antibiotic tolerance and biofilm formation. At high concentrations, (p)ppGpp halts bacterial growth, inhibits protein synthesis and rewires transcription and metabolism. In Escherichia coli  (E. coli ) the (p)ppGpp levels are controlled by two large multi-domain proteins RelA and SpoT, the namesakes of Rel-SpoT Homolog (RSH) protein family. During amino acid starvation, RelA directly senses the acylation status of the A-site tRNA on the ribosome. In the presence of uncharged tRNA, RelA is activated to synthesize (p)ppGpp using ATP and either GDP or GTP as substrates; SpoT opposes the activity of RelA by hydrolyzing the alarmone. The RSH family also includes single domain, monofunctional enzymes: Small Alarmone Synthetases (SASs), which can synthesize (p)ppGpp, and Small Alarmone Hydrolases (SAHs), which can hydrolyze (p)ppGpp. Acting together with ‘long’ RSHs such as RelA and SpoT, these enzymes control the intracellular alarmone levels. Using conservation of genomic neighborhoods analysis of RSH sequences, we have identified several families of SAS factors encoded in conserved bicistronic architectures that are similar to the so-called toxin-antitoxin operons. We experimentally validated five of these SASs as being the toxins (toxSASs) which are neutralized by the products of the six neighboring antitoxin genes. The SAS enzyme from Cellulomonas marina  (C. marina ) FaRel inhibits the growth of E. coli cells by synthesizing alarmones ppGpp and ppApp, which in turn leads to the depletion of cellular ATP and GTP. These toxic effects can be countered by the C. marina SAH antitoxin through degradation of ppGpp and ppApp alarmones.Since (p)ppGpp plays such a crucial role in bacterial virulence and antibiotic tolerance, the (p)ppGpp-mediated signaling has emerged as a target for developing new antibacterials. ppGpp-mimetics are a promising strategy for direct inhibition of RSH enzymes. We tested a targeted chemical library of ppGpp analogs in enzymatic assays with purified E. coli RelA activated by the ribosome. Although the screen has yielded several potent inhibitors, none of them were effective in live bacterial cells. Despite their limited utility as antibacterials, these compounds are useful tools for future structural and biochemical work. We took an alternative approach and developed a High Throughput Screening (HTS) assay which utilized amino acid auxotroph Bacillus subtilis lacking (p)ppGpp. We have performed an HTS screen with a diverse compound library and identified a set of compounds sharing a common 4-(6-(phenoxyl) alkyl)-3,5-dimethyl-1H-pyrazole core as possible stringent response inhibitors. Our follow-up characterization of these compounds as well as reported potential inhibitors — the ppGpp analog Relacin and cationic peptide 1018 — revealed that neither compound is sufficiently specific to warrant further development.