2.4 SOS response in persister cells
The SOS pathway is an inducible DNA damage repair system that is pivotal for bacterial adaptation, pathogenesis, and diversification of population. The SOS response activates systems to carry out adaptation processes to face hostile or even toxic environments. These systems can contribute to growth arrest, DNA repair, repair of replication errors or mobilization of mobile genetic elements (transposons and insertion sequences) [91], [92], [93]. Activation of the SOS response in persister cells could explain the appearance of adaptative mutations, especially in the presence of antibiotics inducing reactive oxygen species (ROS) production leading therefore to DNA damages [94]. Persister could thus be a key stage in the course of genetic resistance development [95]. The SOS response seems to be a key player in persister physiology, for both maintaining DNA integrity and remodeling it [96]. However, DNA damage response is a multifactorial response that appear to be species-specific and may have a major influence on the development of new capabilities of the species [97].
The DNA damages induced by ROS can be produced by antibacterial molecules and leads to an SOS response [98]. This response is mediated by RecA, which binds to single-stranded DNA and protects it from degradation. In many bacteria, LexA is the transcriptional repressor of RecA and of all SOS response genes. It is cleaved by RecA if the latter is bound to a single strand of DNA activating the transcription of SOS genes. In Acinetobacter , the mechanism seems different, as the genome lacks the lexA gene [99], [100]. Indeed, the error-prone reserve polymerase UmuD (polymerase V) is recruited during SOS response. UmuD is repressed by UmuDAb, itself repressed by DdrR. DdrR overexpression represses the DNA repair genes [101].
It was shown that, after 1h tobramycin treatment at 10 × MIC,umuD and ddrR of ATCC 17978 strain were over-expressed [102]. The transcriptomics of ATCC 17978 persisters selected by treatment of 50 × MIC of ciprofloxacin, showed overexpression of different systems, like the DNA repair system, the phenylacetic acid degradation pathway, the leucine catabolism, HicAB toxin-antitoxin system and ROS response (Table1) [63]. Several genes displayed significant changes like pare (A1S_3359); recA (A1S_1962); umuC (A1S_2008); umuD (A1S_1174, A1S_1389); ddrR (A1S_1388); ruvB (A1S_2588);recB (A1S_0356) and repA (A1S_0663). The construction of deletion mutants of recA (A1S_1962) and umuD homolog (A1S_1389) was performed. The umuD mutant formed fewer persister cells than the WT strain, when the recA mutant did not form any persister cells after 3 hour (h) of antibiotic treatment [63]. These results suggested that these proteins (umuD and recA ) are involved in persister formation. It should be also noticed that RecA is involved in resistance to some β-lactam antibiotics and contributes to survival after ultraviolet (UV) treatment, and to virulence in macrophages in vitro and in mice in vivo [103], [104].
Other transcriptomic analyses on ATCC 19606 and two clinical strains treated by 10 mg/L of ciprofloxacin and 10 mg/L of imipenem for 6 h were also performed [105]. They showed similarly that the three strains overexpressed dam, recC, umuD, phoU , and glpD . The methyltransferase Dam, when subcloned under the control of an inducible promoter, positively regulated recC , umuD , phoU , and glpD genes. Consequently, the percentage of persister cells was more abundant in the Dam-overexpressing mutant. The RecC overexpressing-mutant presented also an increased rate of persister cell formation (Table 1) [105].
Antibiotic shock treatment frequently leads to the generation of ROS [106], [107], [108], [109]. As seen previously, ROS may damage DNA leading to the SOS response [96]. However, the bacteria may also activate some other mechanisms to face the ROS stress and maintain the cell integrity. These mechanisms are essential for a rapid adaptation or the bacterial survival. This response is indirectly involved in the state of persister cells [110]. As an example, pyocyanin generates ROS in A. baumannii . It was shown ROS production leads to catalase and Sod increased expression in order to eliminate ROS [111]. Clearance of A. baumannii polymyxin B persister by Rifampicin was shown to be related to a significant down regulation of sodB and therefore to an enhanced generation of ROS (Table 1) [112]. Additionally, it was shown that a SodB A. baumannii mutant was more sensitive to antibiotics and generated less persister cells [112].