# Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria
## 5 Exploitation of TA Systems for the Development of Novel Antibiotic Drugs
A variety of toxin genes could be initially acquired from many known TA operons. However, the use of the bacteriophages has been restricted due to their infections for specific organisms. The most commonly used strategy for the artificial activation of toxins is to use the structural and biochemical information of type II TA proteins to design peptides that inhibit the TA interactions. For example, peptide inhibitors of the _B. antifracis_ PemIK interaction were designed to mimic the _C_-terminal toxin-binding region of the antitoxin [193]. The toxin PemK cleaves single-stranded RNAs, and toxicity is inhibited by the antitoxin PemI. Deletion mutant studies showed that the _C_-terminal fragment of PemI is responsible for PemK binding [194]. The two peptides (LLFQHLTE and RRGYIEMG) block the PemIK interaction and inhibit in vitro ribonuclease activity of the PemK toxin, whereas peptides derived from the _N_-terminal fragment of PemI do not influence the TA interaction [193]. A rationally designed octapeptide (SKIGAWAS) is predicted to form an \(\alpha\)-helix and to occupy the TA binding interface [194]. Consistent with this prediction, this peptide has been shown to inhibit TA complex formation but also decrease the ribonuclease activity of the toxin. However, in order to be candidates of potent antibiotics, a molecule has to not only disrupt the TA interactions but also fully recover the intrinsic activity of the toxin. The latter might be achieved unless a molecule affects the catalytic site of the toxin. An extensive screening of peptides that disrupt the interaction between the antitoxin \(\varepsilon\) and the toxin \(\zeta\), from _S. pyogenes_ plasmid was carried out using bioluminescence resonance energy transfer (BRET) signals between Luc-\(\varepsilon\) and \(\zeta\)-GFP fusion proteins [186]. A considerable number of peptide libraries including over \(4.95\times 10^{7}\) 6-residue peptides,2.74 \(\times\) 10\({}^{4}\) 14-residue \(\beta\)-sheet peptides, and 2.74 \(\times\) 10\({}^{4}\) 17-residue \(\alpha\)-helix peptides were tested for their abilities to prevent TA interactions. Among them, only a few members of the 17-residue library were observed to decrease the BRET signal, indicating that they are able to disrupt the TA interaction. On the basis of the crystal structure of the VapBC complex (Rv0623-Rv0624), three peptides that mimic the TA interface were designed [176]: an 8-residue peptide I from the \(\alpha\)1-helical region of Rv0623 (residues 52-59), a 17-residue peptide II from the \(\alpha\)2-helical region of Rv0624 (residues 14-30), and a nine-residue peptide III from the \(\alpha\)4-helical region of Rv0624 (residues 48-56). How much they increased the ribonuclease activity of the VapC toxin from the non-toxic VapBC complex was evaluated. The recovery of the activity could be explained by the peptide that liberates the toxin from the TA complex. As a result, peptides II and III increased the ribonuclease activity of the VapC toxin more than peptide I. This is an example of the toxin-mimicking peptide that inhibits the TA interaction more strongly than the antitoxin-mimicking peptide. Figure 9: Schematic antibacterial strategies using TA systems. **(A)** Disruptors of TA complex. Direct activation of the toxin is achieved by disruption of the preformed TA complex or prevention of complex formation. The latter can be achieved by either an antitoxin binder or a toxin binder. **(B)** Activation of cellular proteases. A molecule activates cellular proteases, which promotes the degradation of the labile antitoxin protein from the TA complex. **(C)** Inhibition of TA gene transcription. A molecule binds to the promoter of TA operon, inhibiting its transcription. The antitoxin is not replenished, and the remaining antitoxin is degraded. Free toxin acts on cellular targets. **(D)** Inhibition of antitoxin translation. Antisense RNA forms complementary base-paring with antitoxin mRNA, which inhibits antitoxin translation. Free toxin inactivates cellular targets. **(E)** Bacteriophage infection.