The Haemophilus influenzae HipBA toxin-antitoxin system adopts an unusual three-com-ponent regulatory mechanism.

Type II toxin-antitoxin (TA) systems encode two proteins: a toxin that inhibits cell growth and an antitoxin that neutralizes the toxin by direct inter-molecular protein-protein inter-actions. The bacterial HipBA TA system is implicated in persister formation. The Haemophilus influenzae HipBA TA system consists of a HipB antitoxin and a HipA toxin, the latter of which is split into two fragments, and here we investigate this novel three-com-ponent regulatory HipBA system. Structural and functional analysis revealed that HipA N corresponds to the N-ter-minal part of HipA from other bacteria and toxic HipA C is inactivated by HipA N , not HipB. This study will be helpful in understanding the detailed regulatory mechanism of the HipBA N+C system, as well as why it is constructed as a three-com-ponent system.