Balasubramanian Vigneshkumar, Sellegounder Durai, Suman Kundu, Krishnaswamy Balamurugan
Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, India.
Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
J Proteomics. 2016 Aug 11;145:141-152. doi: 10.1016/j.jprot.2016.04.025. Epub 2016 Apr 21.
Caenorhabditis elegans-Pseudomonas aeruginosa infection model is commonly used for pathogenesis studies over the decades. In the present study, upon exposure to the Pseudomonas aeruginosa PAO1, the 2D-PAGE was performed to examine the total proteins differences of C. elegans during the PAO1 infection at different time durations (12-48h). Also, the 2D-DIGE using the cyanine dyes were performed (48h) to identify the differentially regulated proteins against the PAO1 infection. Among the 19 short-listed proteins, 5 proteins were down-regulated and 14 proteins were up-regulated. Eukaryotic elongation factor-2 (EEF-2), a GTP binding protein involves in protein elongation process was down regulated during the pathogen infection. The 2D-PAGE analysis and MS data for the 12 and 24h infections identified the NDK-1 and other essential protein includes, ACS-18, ACT-1, GPD-3, GDH-1 and LBP-6 which are involved in important cellular homeostasis were down regulated. Validation studies using qPCR analysis for eef-2 and other selected genes, western blot analysis for EEF-2 and effect of host translational inhibition studies using Cycloheximide during PAO1 infection suggests that P. aeruginosa systematically restrains the function of host by arresting the expression of EEF-2 and thereby inhibiting protein translational events. Further, in silico analysis revealed the Exotoxin A could directly bind with the host EEF-2 and NDK-1 during the C. elegans- PAO1 interactions.
Model system, C. elegans facilitates the identification of virulence mechanisms during bacterial pathogenesis. Upon infection by the fungal and bacterial pathogens, the C. elegans system induces an array of transcriptional responses, including differential expression of effector/modulator genes that provide safeguard and fight against infection. However, the in-depth knowledge of host response by the pathogen at protein level remains unclear. Much of the studies were carried out only at the transcripts level and scarce reports are available at the protein level for the host-pathogen interaction studies. In order to provide few interesting clues at the protein level, the nematode, C. elegans was infected with the human pathogen P. aeruginosa and the response(s) of host was investigated at the protein level by 2D-DIGE analysis and further validation studies using qPCR and western blotting techniques. Our differential proteomics data suggest that translational inhibition as one of the patterns of pathogenesis in C. elegans during P. aeruginosa infection. Since many of the effectors identified through C. elegans are conserved in other systems including human, our data pave the way for understanding important regulatory pathways involved during bacterial pathogenesis that can be translated into higher eukaryotic organisms.
几十年来，秀丽隐杆线虫-铜绿假单胞菌感染模型常用于发病机制研究。在本研究中，秀丽隐杆线虫暴露于铜绿假单胞菌PAO1后，进行二维聚丙烯酰胺凝胶电泳（2D-PAGE）以检测在不同时间段（12 - 48小时）PAO1感染期间秀丽隐杆线虫的总蛋白差异。此外，还进行了使用花青染料的二维差异凝胶电泳（2D-DIGE）（48小时）以鉴定针对PAO1感染的差异调节蛋白。在19个入围蛋白中，5个蛋白下调，14个蛋白上调。真核延伸因子-2（EEF-2），一种参与蛋白质延伸过程的GTP结合蛋白，在病原体感染期间下调。12小时和24小时感染的二维聚丙烯酰胺凝胶电泳分析和质谱数据确定了NDK-1和其他必需蛋白，包括参与重要细胞内稳态的ACS-18、ACT-1、GPD-3、GDH-1和LBP-6下调。使用qPCR分析eef-2和其他选定基因的验证研究、EEF-2的蛋白质印迹分析以及在PAO1感染期间使用环己酰亚胺进行的宿主翻译抑制研究的效果表明，铜绿假单胞菌通过阻止EEF-2的表达从而抑制蛋白质翻译事件来系统性地抑制宿主功能。此外，计算机分析显示在秀丽隐杆线虫 - PAO1相互作用期间，外毒素A可直接与宿主EEF-2和NDK-1结合。
秀丽隐杆线虫模型系统有助于鉴定细菌发病机制中的毒力机制。在被真菌和细菌病原体感染后，秀丽隐杆线虫系统会引发一系列转录反应，包括效应器/调节基因的差异表达，这些基因提供保护并对抗感染。然而，病原体在蛋白质水平上对宿主反应的深入了解仍不清楚。许多研究仅在转录水平上进行，而关于宿主 - 病原体相互作用研究的蛋白质水平的报道很少。为了在蛋白质水平上提供一些有趣的线索，将线虫秀丽隐杆线虫感染人类病原体铜绿假单胞菌，并通过二维差异凝胶电泳分析以及使用qPCR和蛋白质印迹技术的进一步验证研究在蛋白质水平上研究宿主的反应。我们的差异蛋白质组学数据表明，翻译抑制是秀丽隐杆线虫在铜绿假单胞菌感染期间发病机制的模式之一。由于通过秀丽隐杆线虫鉴定的许多效应器在包括人类在内的其他系统中是保守的，我们的数据为理解细菌发病机制中涉及的重要调节途径铺平了道路，这些途径可以转化为高等真核生物。
