Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

Sabrina Montluc,Alexander Belyy,Undine Mechold,Celia Deville,Louis Renault,Lina Worpenberg,Christophe Velours,Dorothée Raoux-Barbot,Daniel Ladant,Véronique Henriot

doi:10.1371/journal.pone.0206133

Abstract

Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.

Highlights

Several bacterial pathogens produce nucleotidyl cyclase toxins that are delivered to specific target cells of their host where they are activated to synthetize massive amounts of cyclic nucleotides that have key regulatory roles in many cellular processes
We recently discovered that the nucleotidyl cyclase toxin ExoY from Pseudomonas aeruginosa (PaExoY) is activated by actin [6] and showed that this is the case for a rather distant homolog (~38% sequence similarity) of ExoY, VnExoY-like from Vibrio nigripulchritudo, an emerging marine pathogen infecting farmed shrimps in New Caledonia and other regions in the Indo-Pacific [7]
We show that two activated nucleotidyl cyclases (AA-NC), VnExoY and PaExoY, despite having actin as common activator in eukaryotic cells differ significantly: while PaExoY requires F-actin for maximum activation, VnExoY is better activated by G-actin

Summary

Introduction

Several bacterial pathogens produce nucleotidyl cyclase toxins that are delivered to specific target cells of their host where they are activated to synthetize massive amounts of cyclic nucleotides (cNMPs) that have key regulatory roles in many cellular processes. CNMPs subsequently triggers profound alterations of the physiology of the host target cells These enzymes are essentially inactive within their native bacterial host in order to prevent detrimental effects of massive cNMP synthesis and become catalytically active only upon reaching the eukaryotic environment of the host target cells. These toxins are typically activated by interacting with a specific eukaryotic cofactor, usually an abundant protein of their hosts. We further characterized VnExoY from V. nigripulchritudo and showed that in contrast to P. aeruginosa ExoY, this enzyme is preferentially activated by G-actin These two enzymes display different nucleotide substrate and divalent cation specificities. This study indicates that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common mode of action may trigger a much wider range of biological effects on infected cells than initially anticipated

Results and discussion

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Conclusions

Materials and methods