Marcelino Isabel, Colomé-Calls Núria, Holzmuller Philippe, Lisacek Frédérique, Reynaud Yann, Canals Francesc, Vachiéry Nathalie
CIRAD, UMR ASTRE, Petit-Bourg, France.
ASTRE, CIRAD, INRA, Université de Montpellier, Montpellier, France.
Front Microbiol. 2019 Mar 15;10:450. doi: 10.3389/fmicb.2019.00450. eCollection 2019.
Unraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins ( = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir ( = 64/371) and ERGatt ( = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 -glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are -GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on biology, rising interesting questions regarding ER-host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of -GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589.
弄清楚哪些蛋白质和翻译后修饰（PTM）在不同环境中影响细菌的致病性和生理学是一项艰巨的挑战。在此，我们使用基于质谱的分析方法来研究加德尔强毒株（ERGvir）和减毒株（ERGatt）中的蛋白质磷酸化和糖基化，以及它们如何调节生物过程。S/T/Y磷酸化蛋白质组的表征显示，两种菌株共享相同的一组磷酸化蛋白质（=58），其中36%在ERGvir中过表达。酪氨酸磷酸化的比例很高（23%），并且66%的已鉴定肽段是多磷酸化的。糖蛋白质组学揭示了高比例的糖蛋白（ERGvir中为67%），其中一部分糖蛋白是ERGvir特有的（=64/371），还有一部分是ER是ERGatt特有的（=36/343）。这些糖蛋白参与关键的生物过程，如蛋白质、氨基酸和嘌呤生物合成、翻译、毒力、DNA修复和复制。无标记定量分析显示，ERGvir中有31种蛋白质过表达，ERGatt中有8种蛋白质过表达。虽然进一步的PNGase消化分别在ERGvir和ERGatt中可靠地定位了2种和5种N-糖蛋白，但蛋白质印迹法表明许多糖蛋白是O-连接的N-乙酰葡糖胺化的。在这两种变体的磷酸化蛋白质组和糖蛋白质组中都检测到了23种蛋白质。这项工作代表了对加德尔菌生物学中PTM的首次全面评估，提出了关于加德尔菌与宿主相互作用的有趣问题。磷酸化蛋白质组表征表明加德尔菌磷酸化蛋白质参与不同机制的通用性增加。大量的糖蛋白以及缺乏编码糖基转移酶的基因突出了加德尔菌在自身蛋白质糖基化方面对宿主和/或载体细胞机制的依赖性。此外，这些糖蛋白对于与加德尔菌环境变化相互作用和做出反应可能至关重要。O-连接的N-乙酰葡糖胺化和磷酸化之间的PTM串扰可能被用作加德尔菌中的一种主要细胞信号传导机制。由于对加德尔菌蛋白质/蛋白质组及其信号生物学了解甚少，本文给出的结果为进一步通过磷酸化和糖基化事件对加德尔菌蛋白质调控进行假设驱动的探索提供了有用的资源。质谱蛋白质组学数据已存入蛋白质组交换联盟，数据集标识符为PXD012589。
