Comparative study of adenosine 3'-pyrophosphokinase domains of MuF polymorphic toxins.

Polymorphic toxins (PT) are multidomain proteins used for interbacterial competition and pathogenesis. The N-terminal domain of PT specifies the mode of transport and names the family, while the variable C-terminal domain carries the toxic activity, which can be counteracted by immunity proteins that protect the PT-producing bacterium. The MuF family of polymorphic toxins is specifically associated with temperate phages, and our recent work showed that the C-terminal domain of a MuF toxin encoded by a Streptococcus pneumoniae prophage carries adenosine 3'-pyrophosphokinase activity. This type of toxin, which combines a MuF N-terminal domain and an adenosine 3'-pyrophosphokinase C-terminal domain, is called Apk2 for adenosine 3'-pyrophosphokinase family 2. Here, we extend the characterization of this novel family of toxins by providing information on two new members encoded by prophages of Mannheimia haemolytica and Pasteurella multocida. Production of their adenosine 3'-pyrophosphokinase domains (Apk2) in the heterologous host Escherichia coli revealed different levels of toxicity, essentially due to their stability. In vitro assays with the purified M. haemolytica Apk2 domain demonstrated that, identically to that of S. pneumoniae, it exclusively produces (p)ppApp from ATP. The role of immunity proteins and their interchangeability in cross-protection and protein-protein interaction assays was tested. While the immunity proteins that hydrolyse pppApp to ATP are interchangeable, those that inhibit the toxin by protein-protein interaction are mainly active against their intrastrain partner. Overall, this study highlights the conserved features of these enzymatic domains, such as their toxicity, their specific activity toward ATP, and their universal and specific immunities.