Luche Sylvie, Eymard-Vernain Elise, Diemer Hélène, Van Dorsselaer Alain, Rabilloud Thierry, Lelong Cécile
Pro-MD team, Université Joseph Fourier, CEA Grenoble, iRTSV, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UJF, Grenoble, France.
Laboratoire de Spectrométrie de Masse Bio-Organique, IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; CNRS, UMR7178, 67087 Strasbourg, France.
J Proteomics. 2016 Mar 1;135:170-180. doi: 10.1016/j.jprot.2015.07.018. Epub 2015 Jul 26.
Microorganisms, such as bacteria, are one of the first targets of nanoparticles in the environment. In this study, we tested the effect of two nanoparticles, ZnO and TiO2, with the salt ZnSO4 as the control, on the Gram-positive bacterium Bacillus subtilis by 2D gel electrophoresis-based proteomics. Despite a significant effect on viability (LD50), TiO2 NPs had no detectable effect on the proteomic pattern, while ZnO NPs and ZnSO4 significantly modified B. subtilis metabolism. These results allowed us to conclude that the effects of ZnO observed in this work were mainly attributable to Zn dissolution in the culture media. Proteomic analysis highlighted twelve modulated proteins related to central metabolism: MetE and MccB (cysteine metabolism), OdhA, AspB, IolD, AnsB, PdhB and YtsJ (Krebs cycle) and XylA, YqjI, Drm and Tal (pentose phosphate pathway). Biochemical assays, such as free sulfhydryl, CoA-SH and malate dehydrogenase assays corroborated the observed central metabolism reorientation and showed that Zn stress induced oxidative stress, probably as a consequence of thiol chelation stress by Zn ions. The other patterns affected by ZnO and ZnSO4 were the stringent response and the general stress response. Nine proteins involved in or controlled by the stringent response showed a modified expression profile in the presence of ZnO NPs or ZnSO4: YwaC, SigH, YtxH, YtzB, TufA, RplJ, RpsB, PdhB and Mbl. An increase in the ppGpp concentration confirmed the involvement of the stringent response during a Zn stress. All these metabolic reorientations in response to Zn stress were probably the result of complex regulatory mechanisms including at least the stringent response via YwaC.
微生物，如细菌，是环境中纳米颗粒的首要作用目标之一。在本研究中，我们以硫酸锌作为对照，通过基于二维凝胶电泳的蛋白质组学技术，测试了两种纳米颗粒氧化锌（ZnO）和二氧化钛（TiO₂）对革兰氏阳性菌枯草芽孢杆菌的影响。尽管TiO₂纳米颗粒对枯草芽孢杆菌的生存能力有显著影响（半数致死剂量LD₅₀），但对蛋白质组图谱没有可检测到的影响，而ZnO纳米颗粒和硫酸锌显著改变了枯草芽孢杆菌的代谢。这些结果使我们得出结论，本研究中观察到的ZnO的影响主要归因于其在培养基中的溶解。蛋白质组学分析突出了与中心代谢相关的12种调节蛋白：MetE和MccB（半胱氨酸代谢）、OdhA、AspB、IolD、AnsB、PdhB和YtsJ（三羧酸循环）以及XylA、YqjI、Drm和Tal（磷酸戊糖途径）。生化分析，如游离巯基、辅酶A - SH和苹果酸脱氢酶分析，证实了观察到的中心代谢重定向，并表明锌胁迫诱导了氧化应激，这可能是锌离子与巯基螯合应激的结果。受ZnO和硫酸锌影响的其他模式是应急反应和一般应激反应。九种参与应急反应或受其控制的蛋白质在存在ZnO纳米颗粒或硫酸锌的情况下表现出改变的表达谱：YwaC、SigH、YtxH、YtzB、TufA、RplJ、RpsB、PdhB和Mbl。ppGpp浓度的增加证实了锌胁迫期间应急反应的参与。所有这些对锌胁迫的代谢重定向可能是复杂调节机制的结果，至少包括通过YwaC的应急反应。
