Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells.

Frédéric Goormaghtigh,Thibaut Hallaert,Marta Putrinš,Sergo Kasvandik,Tanel Tenson,Andreas Sjödin,Laurence Van Melderen,Nathan Fraikin,Klas Udekwu,Abel Garcia-Pino,Vasili Hauryliuk,Niilo Kaldalu,Gisela Storz

doi:10.1128/mbio.00640-18

Abstract

ABSTRACTPersistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics.

Highlights

IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence
The authors reported that stochastic accumulation of (p)ppGpp was the trigger for degradation of antitoxins resulting in activation of TA systems [34]
A small number of type II persister cells were observed; fluorescence of these cells was comparable to that of the bulk of the population, confirming that there is no direct link between induction of TA systems and persistence to ampicillin

Summary

Introduction

IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity These systems were thought to be pivotal players in persistence until recent developments in the field. An Escherichia coli strain lacking five TA systems (relBE, yefM-yoeB, mazEF, chpB, and dinJ-yafQ) for which the toxins are endoribonucleases, had no survival defects in stress conditions [32] This strain did not show any fitness disadvantage in competition experiments with the wild-type strain. Deletion of the gene encoding the Lon protease, thought to mediate degradation of different antitoxins, had a similar effect While this model gained wide acceptance, several independent follow-up studies questioned its validity [40,41,42,43]. Gerdes lab subsequently proposed that the HipA toxin from the type II hipBA system induces persistence through the activation of the 10 TA systems, reinforcing their role as major effectors of bacterial persistence [44]

Methods

Results

Conclusion