Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella

Abstract

In the search for novel antimicrobial therapeutics, toxin-antitoxin (TA) modules are promising yet underexplored targets for overcoming antibiotic failure. The bacterial toxin Doc has been associated with the persistence of Salmonella in macrophages, enabling its survival upon antibiotic exposure. After developing a novel method to produce the recombinant toxin, we have used antitoxin-mimicking peptides to thoroughly investigate the mechanism by which its cognate antitoxin Phd neutralizes the activity of Doc. We reveal insights into the molecular detail of the Phd–Doc relationship and discriminate antitoxin residues that stabilize the TA complex from those essential for inhibiting the activity of the toxin. Coexpression of Doc and antitoxin peptides in Salmonella was able to counteract the activity of the toxin, confirming our in vitro results with equivalent sequences. Our findings provide key principles for the development of chemical tools to study and therapeutically interrogate this important class of protein–protein interactions.

Stapled Phd Peptides Inhibit Doc Toxin Induced Growth Arrest in Salmonella

“…We previously characterized the C-terminal domain of S. typhimurium Phd antitoxin peptide (Phd STm 52–73 ) as a high affinity inhibitor of Doc STm toxin, exhibiting pM binding affinity and Doc STm inhibition activity comparable to full-length Phd STm 1–73 protein . Based on this activity, the Phd STm 52–73 peptide holds great promise as a Doc STm toxin inhibitor to tackle and study antibiotic persistence in S.…”

“…As a starting point for the generation of cell-permeable Phd STm 52–73 peptides, we used the wild-type Phd 52–73 sequence with N-terminal acetylation and an additional N-terminal tryptophan residue to allow spectrophotometric quantification, as previously described . All peptides were prepared by an automated microwave-assisted solid-phase peptide synthesis (SPPS) using the Fmoc/ t -Bu strategy.…”

“…For modified peptides, 16 – 21 a loss in Doc STm stabilization compared to 1 was observed by thermal shift (Δ T m ≈ 15–20 °C). In addition, the Doc inhibition activity of the peptide analogues was assessed in a phosphorylation assay with recombinant EF-Tu STm and Doc STm and analyzed by dot blot as previously described . Peptide 1 and analogues 16 and 19 fully inhibited EF-Tu STm phosphorylation by Doc STm when present at three times the concentration of Doc STm in the assay (1 μM), while analogues 17 , 18 , 20, and 21 required a higher concentration to achieve full inhibition (Figure C).…”

“…29,30 We have previously carried out a comprehensive characterization of the Phd-Doc interface and demonstrated that antitoxin peptides can effectively mimic the activity of the full length Phd protein both in vitro and when expressed in S. typhimurium. 31 This article is licensed under CC-BY 4 Here, we report the development of stapled Phd peptides capable of the rescue of Doc-induced growth arrest when administered to S. typhimurium (Figure 1). Using the Cterminus from S. typhimurium Phd (Phd STm 52−73 ) with pM affinity for Doc (Doc STm ) as a template, we generated a library of analogues.…”

“…Doc functions as a kinase, phosphorylating the translation elongation factor EF-Tu and subsequently inhibiting protein synthesis. , We have previously carried out a comprehensive characterization of the Phd-Doc interface and demonstrated that antitoxin peptides can effectively mimic the activity of the full length Phd protein both in vitro and when expressed in S. typhimurium …”