# Comparative study of adenosine 3′‐pyrophosphokinase domains of MuF polymorphic toxins

## TL;DR

This study explores a new family of bacterial toxins called Apk2, which produce toxic molecules from ATP and are protected by immunity proteins that vary in their effectiveness.

## Contribution

The paper introduces two new members of the Apk2 toxin family and reveals differences in their toxicity and immunity protein interactions.

## Key findings

- Apk2 toxins from different bacteria produce (p)ppApp from ATP and show varying toxicity levels in E. coli.
- Immunity proteins that hydrolyze pppApp are interchangeable, but those that block the toxin via protein interaction are strain-specific.
- The study highlights conserved enzymatic features and specific immunity mechanisms in Apk2 toxin systems.

## Abstract

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 (Apk2tox) in the heterologous host Escherichia coli revealed different levels of toxicity, essentially due to their stability. In vitro assays with the purified M. haemolytica Apk2tox 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.

With the ultimate goal of understanding the association of toxin‐immunity modules to temperate phages, we characterized toxins from three prophages and examined cross‐protection from immunity proteins. The toxins exhibit adenosine 3′‐pyrophosphokinase activity and are toxic in Escherichia coli. Immunity proteins that interfere via protein association are mainly orthologous to their cognate partners, while those hydrolyzing the reaction product are universal.