The physiological activation and consequences of Toxin-Antitoxin systems in Salmonella

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PROJECT SUMMARY/ABSTRACT Bacteria control their growth in response to environmental challenges and sometimes enter a growth arrested state. Growth-arrested bacteria often show remarkable abilities to survive exposure to antibiotics and are known as antibiotic persisters. These bacterial persisters are thought to contribute to the relapse of many infections and to the worrying burden of antimicrobial resistance. Understanding how bacteria establish this growth arrested state can help to develop better antibiotics. Toxin-Antitoxin (TA) modules are widespread pairs of genes involved in bacterial growth control. They are stress responsive systems that enable bacteria to adapt their growth in response to insults such as attack by phage or host immune defense cells. TA systems encode a non- secreted toxin which inhibits an essential cellular function thereby controlling growth, and an antitoxin that neutralizes the toxin. The antitoxin exerts control over the toxin at two levels, through repression of expression and direct neutralization. It is thought that upon stress, the antitoxin is degraded, on one hand de-repressing expression of the operon, and on the other hand liberating the toxin. However, despite numerous studies on toxin functions, very little information is available on how stresses lead to activation of TA systems, from “de- repression” of the TA operon and liberation of the toxin to actual consequences of the activity of the toxin on the bacteria; and the role of these ubiquitous elements remains disputed. The foundation of the work is our prior demonstration that uptake of Salmonella Typhimurium by macrophages is a natural trigger of expression and activity of each of the TA modules encoded by the bacteria. Using a combination of genetic, biochemical, structural and imaging approaches, we will take advantage of this powerful trigger to study how TA systems of the TacAT group are activated (de-repression in aim 1 and liberation of the toxin in aim 2) and the physiological consequences of the activity of Tac toxins in response to attacks inflicted on bacteria by their environment (intoxication in aim 3 and effects of intoxication in aim 4). The knowledge generated will undoubtedly provide insight on other TA systems beyond the Tac family. In addition, it has the potential to transform our understanding of bacterial growth heterogeneity and the associated phenomenon of antibiotic persistence and serve as a springboard to develop better antibiotics.