2.3 Second Messengers
The second messengers, (p)ppGpp and cyclic dimeric diguanosine monophosphate (c -di-GMP), are main signal molecules, the level of which determines the entry in persister state [69], [70], [71].
(p)ppGpp : Indeed, in E. coli , (p)ppGpp is clearly a major player in induction of persister state [72]. Increased levels of (p)ppGpp induce transcription of RpoS and RpoE [73]. ppGpp inhibits deoxyribonucleic acid (DNA) primase[74], and ribosomal ribonucleic acid (rRNA) transcription [75], deregulates an early step in lipopolysaccharide (LPS) formation [76], and binds to ribonucleic acid (RNA) polymerase [77]. ppGpp could directly inhibit the negative DNA supercoiling [78], and indirectly induces transcription of ribosomal modulation factor (Rmf) and hibernation promotion factor (Hpf), which plays a role in ribosome dimerization (90S and 100S) [79]. In A. baumannii it was shown that the formation of (p)ppGpp, is mediated by RelA (ABUW_3302) (Table1) [80]. RelA is a monofunctional protein only contributing to pppGpp synthesis. Moreover, the reduction of pppGpp into ppGpp is mediated by SpoT, a monofunctional (p)ppGpp hydrolase [81]. TherelA deletion resulted in the formation of 4-fold less persister cells than in WT AB5075 strain under 50 × MIC colistin or 80 × MIC rifampin treatments. The mutant relA underwent also variations of its motility, morphology or colony morphology [80]. In other strains like ATCC 17978, it was shown that a mutant deficient in (p)ppGpp production (ΔA1S _0579 ) is 2- to 4-fold more susceptible to antibiotics, i.e. MICs are reduced by a factor of 1 to 4 compared to wide type (WT) or complemented strain [82]. The authors suggest this susceptibility could be due to the under-expression of efflux transporters (AdeI, AdeJ and AdeK ) and, less obviously, to the over-expression of certain proteins belonging to the transporter systems, such as AetA, AdeB and AdeM [82], [83].
c-di-GMP : It was already shown that this second messenger molecule increases the proportion of persister cells inE. coli [84]. It also influences virulence factors in many bacteria [85], [86], [87]. In P. aeruginosa , HigB decreases the intracellular level of c -di-GMP, which is responsible for the increased expression of the T3SS genes and the repression of biofilm formation [88]. It is worth noting that the enzymes, diguanylate cyclases, involved in c -di-GMP synthesis contain a catalytic site with a GGDEF/EAL motif [89]. In A. baumannii ATCC 17978 strain, several diguanylate cyclases were identified and characterized but their involvement in persister cells has never been studied [90].