Unité de Biochimie des Interactions macromoléculaires, Département de Biologie Structurale et Chimie, CNRS UMR 3528, Institut Pasteur, Paris, France.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, France.
PLoS One. 2018 Nov 12;13(11):e0206133. doi: 10.1371/journal.pone.0206133. eCollection 2018.
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.
几种细菌病原体产生核苷酸环化酶毒素来操纵真核宿主细胞。在宿主细胞内，它们被内源性辅助因子激活，产生高水平的环核苷酸（cNMPs）。铜绿假单胞菌（PaExoY）的 ExoY 毒素和在 Vibrio nigripulchritudo 的 MARTX（多功能自动加工重复毒素）毒素中发现的 ExoY 样模块（VnExoY）具有适度的序列相似性（~38%），但最近都被证明在被递送到真核宿主细胞后被肌动蛋白激活。在这里，我们进一步研究了 V. nigripulchritudo 的 ExoY 样环化酶。我们发现，与需要聚合肌动蛋白（F-actin）才能最大程度激活的 PaExoY 不同，VnExoY 被单体肌动蛋白（G-actin）选择性激活。这两种酶还显示出不同的核苷酸底物和二价阳离子特异性。在存在阳离子 Mg2+的体外条件下，F-actin 激活的 PaExoY 表现出混杂的核苷酸环化酶活性，底物偏好 GTP>ATP≥UTP>CTP，而 G-actin 激活的 VnExoY 对 ATP 表现出强烈的底物偏好，就像博德特氏菌或炭疽杆菌中众所周知的钙调蛋白激活的腺苷酸环化酶毒素一样。这些结果表明，尽管肌动蛋白激活的核苷酸环化酶毒力因子共享共同的激活剂，但它们在感染细胞中可能实际上显示出比最初预期更大的生物学效应的可变性。
