Universidad Autónoma del Estado de Morelos, Morelos, Mexico.
DGIMI, INRA, Université de Montpellier, Montpellier, France.
PLoS One. 2019 Feb 22;14(2):e0212809. doi: 10.1371/journal.pone.0212809. eCollection 2019.
We evaluated the impact of bacterial rhabduscin synthesis on bacterial virulence and phenoloxidase inhibition in a Spodoptera model. We first showed that the rhabduscin cluster of the entomopathogenic bacterium Xenorhabdus nematophila was not necessary for virulence in the larvae of Spodoptera littoralis and Spodoptera frugiperda. Bacteria with mutations affecting the rhabduscin synthesis cluster (ΔisnAB and ΔGT mutants) were as virulent as the wild-type strain. We then developed an assay for measuring phenoloxidase activity in S. frugiperda and assessed the ability of bacterial culture supernatants to inhibit the insect phenoloxidase. Our findings confirm that the X. nematophila rhabduscin cluster is required for the inhibition of S. frugiperda phenoloxidase activity. The X. nematophila ΔisnAB mutant was unable to inhibit phenoloxidase, whereas ΔGT mutants displayed intermediate levels of phenoloxidase inhibition relative to the wild-type strain. The culture supernatants of Escherichia coli and of two entomopathogenic bacteria, Serratia entomophila and Xenorhabdus poinarii, were unable to inhibit S. frugiperda phenoloxidase activity. Heterologous expression of the X. nematophila rhabduscin cluster in these three strains was sufficient to restore inhibition. Interestingly, we observed pseudogenization of the X. poinarii rhabduscin gene cluster via the insertion of a 120 bp element into the isnA promoter. The inhibition of phenoloxidase activity by X. poinarii culture supernatants was restored by expression of the X. poinarii rhabduscin cluster under the control of an inducible Ptet promoter, consistent with recent pseudogenization. This study paves the way for advances in our understanding of the virulence of several entomopathogenic bacteria in non-model insects, such as the new invasive S. frugiperda species in Africa.
我们评估了细菌 rhabduscin 合成对 Spodoptera 模型中细菌毒力和酚氧化酶抑制的影响。我们首先表明，昆虫病原细菌 Xenorhabdus nematophila 的 rhabduscin 簇对于 Spodoptera littoralis 和 Spodoptera frugiperda 幼虫的毒力不是必需的。影响 rhabduscin 合成簇的突变细菌（ΔisnAB 和 ΔGT 突变体）与野生型菌株一样具有毒力。然后，我们开发了一种用于测量 S. frugiperda 酚氧化酶活性的测定法，并评估了细菌培养上清液抑制昆虫酚氧化酶的能力。我们的发现证实，X. nematophila 的 rhabduscin 簇是抑制 S. frugiperda 酚氧化酶活性所必需的。X. nematophila ΔisnAB 突变体无法抑制酚氧化酶，而 ΔGT 突变体相对于野生型菌株表现出中等水平的酚氧化酶抑制。大肠杆菌和两种昆虫病原细菌，Serratia entomophila 和 Xenorhabdus poinarii 的培养上清液均不能抑制 S. frugiperda 酚氧化酶活性。在这三种菌株中异源表达 X. nematophila rhabduscin 簇足以恢复抑制作用。有趣的是，我们观察到 Xenorhabdus poinarii rhabduscin 基因簇通过插入 isnA 启动子中的 120 bp 元件而发生假基因化。X. poinarii 培养上清液对酚氧化酶活性的抑制作用通过在诱导型 Ptet 启动子的控制下表达 X. poinarii rhabduscin 簇得以恢复，这与最近的假基因化一致。这项研究为我们在非模式昆虫中理解几种昆虫病原细菌的毒力奠定了基础，例如非洲新入侵的 S. frugiperda 物种。
