# TisB enables antibiotic tolerance in Salmonella by preventing prophage induction through ATP depletion

## TL;DR

A bacterial toxin called TisB helps Salmonella survive antibiotics by lowering ATP levels, which prevents prophage activation and reduces cell death.

## Contribution

This study reveals that TisB-mediated ATP depletion prevents prophage induction, increasing antibiotic tolerance in Salmonella.

## Key findings

- Deleting tisAB increases prophage induction and reduces persister cell survival after ciprofloxacin treatment.
- Low ATP levels prevent prophage activation, leading to higher bacterial survival during antibiotic treatment.
- The recovery phase after antibiotic removal is crucial for prophage induction in Salmonella.

## Abstract

Persisters are phenotypically antibiotic-tolerant cells which can survive antibiotic exposure without acquiring antibiotic resistance. A proposed important factor in persistence is low intracellular ATP levels, which are thought to reduce the activity of antibiotic targets. However, previous studies demonstrated that persisters have comparable DNA damage as drug-sensitive bacteria after fluoroquinolone treatment. Furthermore, recent studies reported that endogenous prophages can reduce levels of antibiotic persistence in Salmonella after fluoroquinolone treatment. In this study, we examined prophage induction and persister cell survival of a prophage-free variant of Salmonella Typhimurium and strains harbouring a deletion of the tisAB genes, with tisB encoding the toxin from the tisB/istR-1 toxin-antitoxin system, known to reduce the intracellular ATP concentration. Treatment of the prophage-free variant with ciprofloxacin resulted in reduced killing and increased persistence as compared to the wild type. In addition, prophage induction and prophage mediated killing was significantly increased after deletion of tisAB following ciprofloxacin treatment. We also demonstrate that the recovery phase following the removal of ciprofloxacin, is crucial for the induction of endogenous prophages. Our results suggest that ATP-dependent prophage activation plays a significant role in DNA damage-mediated killing of bacteria. Low ATP levels can dampen the induction of prophages and increase the fraction of bacterial survivors after ciprofloxacin treatment.

Antibiotic persistence comprises drug-tolerant bacteria that can survive treatment with antibacterial agents, despite lacking classical genetic resistance mechanisms. Therefore, persisters are clinically relevant because they can lead to treatment failures and chronic infections. Additionally, antibiotic persistence facilitates the evolution of resistance through genetic mutations. Persisters are triggered by a lack of nutrients, bacterial toxins, low ATP levels, or other stress responses that shut down bacterial metabolism. However, the involvement of prophages, viruses that integrate into bacterial chromosomes, is less well understood. In this study, we tested a tisAB deletion in Salmonella Typhimurium and examined persister cell formation following treatment with the DNA-damaging drug ciprofloxacin. TisB is a bacterial toxin that increases the influx of protons across the inner bacterial membrane into the cytosol, causing ATP depletion. We demonstrate that the deletion of tisAB increases prophage induction and bacterial killing, leading to a reduced persister cell fraction. The tisAB mutant is unable to down regulate its ATP concentration after exposure to ciprofloxacin, which in turn allows for stronger binding of RecA to single-stranded DNA, the activator of both the SOS response and prophage induction.