Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in .

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the complex, with 50 modules present in the genome. In type IIA modules the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins also bind to promoter region sequences and repress expression of the operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of - operon transcription would result in increased free VapC in the cell. strains expressing constructs containing a phosphoablative mutation resulted in lower toxicity compared to a strain expressing native , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation, potentially in response to extracytoplasmic as well as intracellular signals.