# Emerging Roles of Toxin-Antitoxin Modules in Bacterial Pathogenesis
## 5 Persistence Emerges in Response to the Activation of Diverse TA Modules  
### Toxin Activity and Persister Cell Formation in E. coli  
Persistence represents a phenotype exhibited by a small fraction of a bacterial population that temporarily enters a dormant state characterized by reduction of growth rates and metabolic activity. Entry into dormancy allows cells to survive unfavorable conditions and to return to the active state when environmental parameters improve. Most antibiotics, as well as many other environmental threats, affect only growing and dividing cells, hence persisters are more protected from killing. It was demonstrated that persisters can arise either stochastically as a result of single cell specific fluctuations in gene expression [89] or in response to a variety of environmental conditions including starvation, oxidative stress, SOS response, carbon source transition and the presence of antibiotics [90; 91; 92; 93]. The contribution of individual TA genes to persistence is difficult to assess because of the functional redundancy of the multiple TA modules encoded within most bacterial chromosomes. However, it has been established unequivocally that TA systems play important roles in persister cell formation [63; 64; 93; 94; 95]. Transcriptomic analysis of _E. coli_ population fractions enriched for persisters revealed that these cells showed significantly increased levels of TA mRNAs [96; 97]. Moreover, persistence can be induced by overproduction of toxins encoded by TA loci and the stimulation of antitoxin gene transcription can reverse this effect [96; 97; 98; 99; 100; 101]. Accordingly, successive deletion of ten endoribonuclease coding TA systems (RelE, YoeB, HigB, YhaV, YafO, YafQ that cleave mRNA positioned at the ribosomal A site, and MazF, ChpB, MqsR, and HicA that cleave RNA in a site specific manner independent of the ribosome) from the _E. coli_ chromosome (Figure 2) produced an additive effect on persister formation, finally reducing persistence more than 100-fold compared to the wild-type strain [90]. At least five TA loci need to be knocked out in _E. coli_ before a significant effect on persister frequency was observed [90]. These results illustrated that the formation of at least 99% of persisters in the exponentially growing culture of _E. coli_ depends on the TA loci, whereas less than 1% of the persisters arise from random fluctuations in the growth rates of single cells [102]. In support of these results, overexpression of Lon, the protease that degrades most antiotoxins, enhanced toxicity and thereby increased the fraction of persisters, whereas the absence of Lon decreased persister formation. The only known activator of Lon protease is inorganic polyphosphate (poly(P)) whose level is influenced by the intracellular concentrations of the stringent response alarmome (p)ppGpp. Thus, (p)ppGpp, which is produced in response to many different types of stresses, induces persister formation by activating TA modules through poly(P) and Lon protease function [103]. A model explaining more specifically the mechanism by which (p)ppGpp and the HipBA type II system mediate persistence was elaborated recently [45; 93; 104]. Here, free HipA toxin inactivates glutamyl-tRNA synthetase (GltX) by phosphorylation which increases the rate of uncharged tRNA\({}^{\text{Glu}}\) loaded at the A site of the ribosome [45; 105]. RelA-dependent (p)ppGpp production is triggered as a result. (p)ppGpp competitively blocks exopolynophasthase which is the enzyme that degrades poly(P). In turn, poly(P) is synthesized by polyphosphate kinase and activates Lon to degrade type II antiotoxins, including HipB. In these conditions the cognate toxin endoribonucleases are liberated causing inhibition of translation and cell growth thereby inducing persistence [45; 95; 104] (Figure 4). The rate of global cellular translation is low in the persistent state, which favors a low antitoxin-toxin ratio due to the instability of the former.