Functional analysis of cysteine residues of the Hok/Gef type I toxins in Escherichia coli.

The Hok/Gef family consists of structurally similar, single-span membrane peptides that all contain a positively charged N-terminal domain, an α-helix and a periplasmic C-terminal domain. Hok/Gef peptides have previously been described to play distinct physiological roles. Indeed, while HokB has been implicated in bacterial persistence, other members of the Hok/Gef family are known to induce cell lysis. However, the generalizability of previously published studies is problematic, as they have all used different expression systems. Therefore, we conducted a systematic study of the nine Hok/Gef peptides of Escherichia coli. We observed rapid cell death following expression of hokA, hokC, hokD, hokE, pndA1, hok or srnB, while expression of hokB or pndA2 does not result in cell lysis. A remarkable feature of Hok/Gef peptides is the presence of conserved periplasmic tyrosine and/or cysteine residues. For the HokB peptide, one of these residues has previously been implicated in intermolecular dimerization, which is essential for HokB to exert its role in persistence. To assess the role of the periplasmic cysteine and tyrosine residues in other Hok/Gef peptides and to decipher whether these residues determine peptide toxicity, an array of substitution mutants were constructed. We found that these residues are important activators of toxicity for Hok, HokA and HokE peptides. Despite the loss of the cell killing phenotype in HokS31_Y48, HokAS29_S46 and HokES29_Y46, these peptides do not exert a persister phenotype. More research is needed to fully comprehend why HokB is the sole peptide of the Hok/Gef family that mediates persistence.

HokB Monomerization and Membrane Repolarization Control Persister Awakening.

Every bacterial population harbors a small subpopulation of so-called persisters that are transiently antibiotic tolerant. These persisters are associated with the recalcitrance of chronic infections because they can recolonize the host after antibiotic removal. Although several effectors have been described to induce persistence, persister cell awakening is poorly understood. We previously reported that the toxin HokB induces persistence via pore formation, resulting in membrane depolarization and ATP leakage. We now delineate mechanisms responsible for the awakening of HokB-induced persisters. We show that HokB dimerization by the oxidoreductase DsbA is essential for pore formation and peptide stability. Pores are disassembled via DsbC-mediated monomerization, which targets HokB for DegQ-mediated degradation. Finally, pore disassembly allows membrane repolarization by the electron transport chain, supporting cells to resume growth. These results provide a detailed view of both the formation and awakening of HokB-induced persister cells.