# Recent Advances in Bacterial Persistence Mechanisms
## 3 Research on the Mechanism of Bacterial Persistence
### Toxin-Antitoxin Modules  
Bacterial toxin-antitoxin (TA) modules extensively exist in a variety of cellular processes [27], and are composed of a toxin protein that inhibits cell growth by interfering with important metabolic activities and an antitoxin that can protect cells from toxins [3; 28].  
Figure 2: The fortress model. When antibiotics, immune cells or other potential environment stress start to stimulate the surface of the biofilm, the outer layer of bacteria will send signals to the bacteria inside to be alert to the bactericidal attack. The gradient of nutrition and oxygen will also contribute to the persister dormancy.  
Some experiments have demonstrated that breaking the balance between the toxin and antitoxin would lead bacterial growth to a stagnant state, and then weaken the harm of antibiotics or other unfavorable circumstances, which could facilitate the survival of bacteria [13; 29]. Environmental stress can induce a graded and controlled activation of toxins to help bacteria enter a transient dormancy state, which is the basis for surviving high-concentration antibiotics [30]. For example, the stress inside the host macrophages is more severe than the nutrtitious culture medium, providing more triggers for the TA modules' activation [2]. To date, TA systems can be divided into eight types based on the properties and working modes of antitoxin, named type I to type VIII, following the discovery order. Type I antitoxins are small RNAs that inhibit the cognate toxin's expression by silencing their transcript. Type II antitoxins are proteins that neutralize the cognate toxin by binding to them and forming tight complexes [31]. While type I TA modules were the first to be discovered, type II are the most abundant and diverse, occurring in the majority of bacteria and some archaea. Since the discovery of type I and type II TA modules on bacterial chromosomes, they have been recognized as beneficial factors in the face of stressful conditions including antibiotic persistence [17]. For type I TA systems, high persistence was reported to be positively correlated with the levels of ObgE and HokB. Obg is a conserved GTPase that plays an important role at the crossroads of the major cellular processes of translation and DNA replication, which are considered to be repressed in dormant persister cells. HokB, a toxin encoded by a type I toxin-antitoxin module, can provoke a collapse in the membrane potential [32]. The gene high persistence protein A (_hipA_)-encoding type II toxin was firstly discovered in _Escherichia coli_ (_E. coli_), whose corresponding antitoxin is HipB [33; 34]. It was reported that strains carrying hipA7 mutants produce persisters at a frequency of 1% compared to 10-5 in wild-type strains when exposed to ampicillin [35]. As the bacterial cell density increases, hipA7 strains also produce persistent cells at an increasing frequency. In addition, the deficiencies of more than five type II TA modules in _E. coli_ would decrease persister formation during the exponential phase [36]. Beside the mutations on TA module components, the expression of TA modules in enriched _E. coli_ persisters was obviously upregulated compared with normal growing bacteria [33; 36]. Furthermore, the Lon protease, a kind of ATPase in _E. coli_, is required for the degradation of the antitoxin, and results in the accumulation of toxins to promote persister formation [29].  
Current studies have been focused on Gram-negative bacteria, especially _E. coli_, though the study on Gram-positive bacteria is not clear. There is evidence that the absence of the TA module in _Staphylococcus aureus_ (_S. aureus_) would not affect the persister level [37]. Nonetheless, it was proposed that TA modules in Gram-positive bacteria work similarly to those in Gram-negative bacteria, and take part in the host's stress response [38].