The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage

Abstract

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coli via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB.

Chromosomally Encoded hok-sok Toxin-Antitoxin System in the Fire Blight Pathogen Erwinia amylovora: Identification and Functional Characterization

“…Interestingly, we also observed membrane blebbing at the division point after hok was induced. Membrane blebbing is a universal phenomenon in bacteria in response to disruption of the plasma membrane (46,47) and suggests that E. amylovora Hok, like Hok homologs from other species, may function through membrane pore formation (16,17). The rapid formation of a cell membrane near the division plane might make this location more vulnerable to the damage of Hok, consistent with this location as the site of visible blebbing.…”

“…While some type I TA toxins cleave nucleic acids in the cytoplasm (e.g., SymE and RalR), Hok and the toxin TisB are membrane associated (15). These toxins functionally resemble pore-forming phage holin proteins, and their expression results in the collapse of membrane potential and leakage of intracellular ATP (9,16,17). In addition to a role in postsegregational killing, Hok and TisB are thought to contribute to bacteriophage exclusion or to antibiotic persistence, a temporary dormant state conferring tolerance to antibiotics (18)(19)(20).…”

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

“…Critically, the persister resuscitation work related with HokB is based on non‐physiological levels of toxin from overproduction studies. [ 38,39 ] In addition, there is little convincing proof that HokB is related to persistence in wild‐type cells since deleting hokB has no effect on persistence. [ 40 ] Furthermore, HokB was identified as related to persistence based on work with the GTPase Obg; however, reduction in persistence based on reducing Obg levels occurs without HokB, [ 40 ] and production of Obg causes a likely inconsequential increase in the induction of hokB (1.7‐fold).…”

Activation of metabolic and stress responses during subtoxic expression of the type I toxin hok in Erwinia amylovora

“…Plasmid expression of the hok-sok locus also increased T4 bacteriophage exclusion in E. coli (18). Interestingly, despite its role in compromising membrane integrity, moderate hokB expression was observed to increase metabolic activity in E. coli, determined via a uorescent redox sensor (10).…”

“…A high induction level of the toxin genes hok or tisB causes drastic cell death of E. coli cells, accompanied by collapse of the proton motive force (PMF) (7)(8)(9). The gene products of both hokB and tisB form membrane pores in Escherichia coli (8,10) and lead to leakage of cellular ATP during moderate (10) or high-level (7) induction of the toxin genes. The PMF, the proton gradient generated via oxidation of NADH and FADH 2 , is required to generate ATP through ATP synthase, as well as to power membrane-localized cell machinery, such as the agellum (11,12).…”