Mechanism and stability of type II bacterial toxin‐antitoxin complexes and the role of regulated antitoxin proteolysis that releases toxin

Abstract

Antibiotic resistance is the most prominent threat facing modern medicine and is unlikely to be resolved by novel antibiotics or reduction of improper antibiotic use. New strategies to combat antibiotic resistance by targeting novel bacterial pathways are necessary. One emerging approach focuses on understanding how bacteria adapt to diverse stresses by the inhibition of growth and transition to a dormant state. These bacteria, termed ‘persisters’, tolerate antibiotics without genetic mutations. This phenotype is triggered by the stringent response whereby transcriptional reprogramming and translational inhibition occurs. A class of ubiquitous bacterial gene pairs called toxin‐antitoxin complexes (TAs) undergo changes during the stringent response that activate the toxin component. Normally antitoxins bind and sequester their cognate toxins to function as autorepressors. In stress conditions, antitoxins are degraded by cellular proteases which frees toxins to inhibit growth until stress passes. We and others demonstrated that proteases recognize C termini of antitoxins. Many studies have focused on identifying toxin targets, yet the stability of TAs and how this influences proteolysis is unknown. Given the structural diversity of antitoxins, it is also unclear how proteases recognize antitoxins or if a common mechanism exists to describe all antitoxin proteolysis. This is an important question as proteolysis is the main step in the release of toxin and the initiation of bacterial persistence. Here, we examine the regulation of E. colitype II TAs DinJ‐YafQ, RelB‐RelE, and YefM‐YoeB. The structures of these pairs are known and demonstrate differences in how DinJ and RelB antitoxins interact with their toxins as compared to YefM. Each pair also forms higher ordered complexes that are differentially regulated. We designed C‐terminal truncations and measured their impact on toxin release and antitoxin stability. We find that DinJ and RelB share common structural motifs at their C termini that are important for toxin sequestration. YefM lacks this motif and its C‐terminal plays no role in toxin sequestration. Further, we explore the role of proteases, these motifs, and effects of diverse stress in targeted antitoxin proteolysis. This work identifies features of antitoxins that may recruit proteases and how proteolysis is a major underlying component of stress allowing bacteria to transition to antibiotic persistent states.Support or Funding InformationThe Dunham laboratory thanks the following sources for funding: NSF CAREER MCB 0953714, NIH NIGMS R01GM072528. Dr. Dunham is a Burroughs Welcome Investigator in the Pathogenesis of Infectious Diseases. Ian Pavelich is an ARCS awardee.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.