# Recent Advances in Bacterial Persistence Mechanisms
## 3 Research on the Mechanism of Bacterial Persistence
### ppGpp and Stringent Response  
If cells encountered amino acid starvation, protein synthesis and other metabolic activities would be closed. This phenomenon is called the stringent response, a type of conservative and global transcriptomic adaptation to environmental pressure [39]. Many studies have confirmed that the stringent response can facilitate persister formation by lapsing into dormancy [7; 40; 41]. The small molecule guanosine tetraphosphate (ppGpp), controlled by Rel/Spo homolog and small alarmon synthetase proteins, is the central molecule in the stringent response [41; 42]. In many biological and animal models, the bacterial mutants that cannot produce ppGpp usually show a reduced ability of persister formation [25; 43; 44; 45]. In the stationary phase, the elevated recruitment of ppGpp to Obg GTPase results in the excessive binding of Obg to the 50S ribosome subunit, which reduces the levels of active 70S ribosomes and thereby regulates cellular functions and participates in various stress adaptions like the stringent response [32; 46]. In persisters, Obg induces the expression of HokB, a membrane-targeted type I toxin, which can damage proton motion and block ATP synthesis, leading to dormancy [32]. Recent evidence has put an emphasis on ppGpp instead of TA modules in persister formation [47]. The steady state of ppGpp is maintained by ReIA and SpoT, while the former is a synthase, and the latter is a hydrolase with weak synthetic activity [48; 49]. It has been proven that ciprofloxacin, cefepime, colistin, and amikacin can upregulate the expression of the relA gene to help persister formation [50]. However, without the presence of ppGpp, such as relA knockout mutants, persisters still exist [51], suggesting that the mechanism needs further investigation. PhoU is another persistence-related factor. PhoU homologs have been identified as phosphate-specific transport system accessory proteins that respond to the environmental Pi level. Deletion of the PhoU1 and PhoU2 in _S. aureus_ caused an increase in both persistence and bacterial virulence [52]. In _P. aeruginosa_, a phoU mutant showed increased levels of intracellular ppGpp, affected antibiotic susceptibility, and decreased growth rate [53]. Interestingly, biofilm formation was not affected by the phoU mutation.  
During the stringent response, the accumulation of ppGpp results in changes in multiple metabolic pathways, involving DNA replication, nucleotide synthesis, transcription, ribosome maturation, and lipid metabolism. For example, the stringent response decreases the initiation rate of DNA replication in _E. coli_[54], and controls 100S ribosome formation by transcriptional regulation in multiple species [55]. rRNA synthesis and genes involved in the metabolism of macromolecules, such as phospholipids, are generally repressed, while the transcription of nutrient transporters is increased to overcome nutrient limitations [56]. In addition, due to the decreased level of lysine during fatty acid starvation, RelA is distinctly activated and induces the synthesis of ppGpp [57].  
To explore the mechanism of how the stringent response increases bacterial cytotoxicity, [PERSON] et al. conducted transposon sequencing and RNA sequencing analyses on _Streptococcus agalacticiae_ (GBS) with human whole blood [39]. Except in the capsular polysaccharide genes, relA was identified to be essential for GBS survival in blood. In addition to promoting the persistence of GBS in host blood, the activation of a stringent response by relA can also enhance the expression of \(\beta\)-hemolysin/cytolysin, a toxin required for GBS virulence [58] through an arginine-mediated metabolism pathway. At present, a small molecule inhibitor aimed at the stringent response has been developed as a new type of antibacterial, and it has demonstrated the corresponding therapeutic effects in pre-clinical tests [59; 60].