DNA damage and evolution of Salmonella antibiotic persisters during infections

Introduction
Antibiotic failure represents a major health concern. The recalcitrant nature of many bacterial infections is thought to be due in part to the presence of persister cells. Persisters have been reported in all major pathogens and are key players in the relapse of bacterial infections. For example, in patients with invasive non-typhoidal Salmonella Typhimurium disease, approximately 78% of recurrent infection was caused by the same strain responsible for the initial disease (Okoro et al, 2012). Moreover, high levels of persisters have been observed in late isolates of Pseudomonas aeruginosa from lung infections in patients with chronic cystic fibrosis (Mulcahy et al, 2010). Unlike inherited resistance, persistence refers to a transient phenotypic state in which a slow-growing subpopulation of bacteria becomes tolerant to antibiotic treatment (Fisher, Gollan & Helaine).
Formation and survival of persisters
Formation of persisters has been shown to be triggered by several stress conditions such as nutrient starvation (Germain, et al, 2015); diauxic carbon-source shifts (Amato & Brynildsen, 2013), pH shift (Helaine et al, 2014) or DNA damage (Dorr et al, 2009). There is also correlation between the activity of toxin-antitoxin (TA) modules and antibiotic persistence (Moyed et al, 1986). Although, persister formation has been extensively studied in laboratory conditions, the ability of these cells to sustain a non-growing state during infection is overlooked. However, in physiologically relevant conditions, infection with intracellular pathogens such as Salmonella Typhimurium and Mycobacterium Tuberculosis results in high numbers of persisters able to survive the macrophage environment (Mouton et al, 2016; Helaine et al; 2014). Persisters can also survive in lung biofilms of patients with cystic fibrosis (Spoering & Lewis, 2001; Moker, Dean & Tao, 2010). Therefore, the mechanism of survival of the persister subpopulation in these environments is important to understand from a clinical point of view.
Evolution of persistence
In vitro studies in E. coli have shown that intermittent antibiotic stress can lead to evolution of antibiotic tolerance (Van den Bergh, et al, 2016). Unlike persistence, which refers to a phenotypic switch in a subpopulation of cells, tolerance is described as a decrease in antibiotic susceptibility at a whole population level, which can be genetically acquired or not (Balaban, 2016). The evolved strains had mutations that rendered them tolerant, rather than resistant to cyclic stress. This suggests that bacteria can adapt to antibiotic treatment through mechanisms other than resistance. Moreover, it was also suggested that evolution of tolerance can aid the subsequent development of resistant mutations (Fridman et al, 2014; Levin-Reisman et al ,2017).
Future objectives:
1. The importance of DNA damage response during persistence
Upon internalization, Salmonella are exposed to the antimicrobial environment of the macrophages. These host immune cells attack engulfed bacteria by generating high levels of oxidative stress, potentially inducing bacterial DNA damage (Burton, et al, 2014). Bacterial cells respond to DNA damage by activating the SOS response, which has been correlated with increased level of persistence. Studies in E. coli have shown that induction of the SOS response following treatment with fluoroquinolones is required for the formation of persisters (Dorr, et al, 2009; Volzing & Brynildsen, 2015).

We will use a library of Salmonella mutants lacking 21 proteins involved in different DNA repair pathways to investigate their ability to survive in a non-growing antibiotic tolerant state while in the stressful macrophage environment. Our aim is to identify DNA repair pathways involved in persistence and test their role in survival of persisters both in vitro following antibiotic treatment or ex vivo during infection of primary bone marrow derive