# Cellular Self-Digestion and Persistence in Bacteria
## 7 Concluding Remarks  
Persistence and self-digestion (or autophagy), which appear to be evolutionarily conserved phenomena, are observed in many prokaryotic and eukaryotic cell types. These processes allow organisms to survive in undesirable environmental conditions, leading to formation of persister cells. Critically, mapping a self-digestion-mediated persistence mechanism from its initial exogenous stress trigger, through its signal transduction, to the source of antibiotic tolerance, may provide us an opportunity to categorize previously identified mechanisms within one complex network. Such a strategy may also uncover novel antipersister therapeutic approaches, as inhibition of intracellular degradation is known to reduce persister formation [234; 245; 55]. Conversely, stimulating self-digestion might also be useful as an alternative antipersister strategy, due to the fact that enhanced intracellular degradation can also be detrimental to persister cells [27; 54]. However, the study of cellular self-digestion mechanisms is challenging, and a number of critical questions remain to be addressed, some of which are as follows:  
1. If a proposed mechanism is essential for persister formation and survival, genetically perturbing the mechanism should eliminate persisters or reduce their levels; however, this method may not be ideal for redundant systems. Self-digestion-mediated persistence is potentially a collective effect of many different degradative enzymes, which makes it difficult to test using conventional methods. One way to investigate these mechanisms is to perform single-cell analysis. With the use of antibiotic treatments and fluorescent reporters for degradative enzymes, a correlation between persistence and the enzymes expression levels can be performed in cell populations where self-digestion is significantly upregulated (e.g., late-stationary-phase cultures). 2. Although [PERSON] et al. claim that persisters and \"viable but non-culturable\" (VBNC) cells represent the same phenotypes [54], the VBNC state is thought to be a transitory phase on the spectrum between persistence and cell death [292; 293]. While persister cells can exit from persistence state (stochastically or deterministically) and colonize, the resuscitation of VBNC cells is rarely observed [235; 293; 294; 35]. In fact, bacteria associated with asymptomatic infections may be in a non-replicating or slowly replicating state and cannot be easily cultured in vitro [295; 296; 297], and this \"viable but non-culturable\" state observed in pathogenic bacteria has long been known [296]. Further, a number of independent groups have shown antibiotic-treated cultures contain many more VBNC cells than persisters [235; 37; 298; 35]. If persisters and VBNC cells represent two distinct phenotypes on the live- and dead-cell spectrum, then, a threshold level of intracellular degradation may play a critical role in the phenotypic switch between persistence and the VBNC state, which remains to be validated. 3. Persister metabolism is a controversial topic, reflecting the complexity and diversity of persister cell formation, survival, and resuscitation mechanisms, as well as the influence of culture conditions [31; 61; 299]. Although persisters are mostly non-growing cells [33; 300; 301; 302], and their metabolism is generally lower than that of exponentially growing cells [13; 37; 102; 233; 103], these phenotypes might be at a metabolic steady state, providing energy molecules necessary for their survival [237; 239]. Although it is well established that autophagy plays a crucial role in the metabolism of drug-tolerant cancer cells, it remains to be determined whether this is also true for bacterial persisters. 4. The levels of global regulators, such as Rpos, ppGpp, and cAMP/Crp, are significantly altered in cells during their transition to stationary phase [110; 116; 117; 117; 126; 144; 147].