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

Abstract

Persistence 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.

Plausible role of bacterial toxin–antitoxin system in persister cell formation and elimination

“…Using this modified protocol, although the importance of (p)ppGpp and Lon protein in E. coli persister generation could be confirmed, the persister forming role of TA genes was found invalid. That TA plays negligible role in mediating persister formation was further confirmed by (Goormaghtigh et al, ) using independently constructed strains for E. coli and few fluorescent reporters at population as well as single cell level.…”

Toxin/Antitoxin System Paradigms: Toxins Bound to Antitoxins Are Not Likely Activated by Preferential Antitoxin Degradation

“…Similarly, evidence based on the overproduction of a non‐toxic toxin such as HipA7 is not convincing for establishing a link between persistence and TA systems. [ 31 ] Critically, several reports have shown that inactivation of 10 TA systems (but not including HipA) has no effect on the persister level of Escherichia coli [ 32–34 ] and deletion of 12 TA systems in Salmonella enterica has no effect on persistence. [ 35 ] Notably, these studies are imperfect in proving persistence is not related to TA systems since E. coli has at least about 40 TA systems [ 24 ] which are interconnected (e.g., toxin MqsR activates toxin GhoT as part of a cascade [ 5 ] ) so only a small percentage of the total number of TA systems were deleted.…”

Stress induces the transcription of toxin-antitoxin systems but does not activate toxin

“…To test this hypothesis, we used a recently constructed ∆10TA strain in which each of 10 type II TA systems in E. coli MG1655 have been deleted to examine growth following a stress (Goeders et al, 2013;Goormaghtigh et al, 2018). This strain was verified as lacking the prophage contaminant found in a different, prior version of this strain.…”

“…For growth experiments, the wild type used was the parental strain to the ∆10TA strain obtained from L. Van Melderen (Goormaghtigh et al, 2018). Cells were grown to OD 600 ~ 0.3 and then stresses were applied as described above.…”

“…They have been suggested to promote stress tolerance, genome stability, persister cell formation, and programmed cell death, but the evidence supporting these functions is often limited, speculative, or controversial (Christensen et al, 2003;Gerdes and Maisonneuve, 2012;Harms et al, 2016; protease to degrade antitoxins at an accelerated rate, thereby freeing toxins to promote persister cell formation (Maisonneuve et al, 2013). However, much of this work was retracted when it came to light that the D10TA strain had acquired multiple f80 prophage insertions that were responsible for the persister phenotypes observed (Goormaghtigh et al, 2018;Harms et al, 2017).…”