Characterization of Entamoeba histolytica adapted to auranofin by combined transcriptomics and redox proteomics. Characterization of Entamoeba histolytica adapted to auranofin by combined transcriptomics and redox proteomics

Purpose: Transcriptome characterization of Entamoeba histolytica trophozoites adapted to Auranofin Total RNA was extracted from control trophozoites (WT) and AFAT using the TRI reagent kit, according to the manufacturer instructions (Sigma-Aldrich USA). 6 RNAseq libraries were produced according to manufacturer protocol (NEBNext UltraII Directional RNA Library Prep Kit for Illumina, cat no. E7760) using 800ng total RNA. mRNAs pull-up was performed using Magnetic Isolation Module (NEB, cat no. E7490). All libraries were mixed into a single tube with equal molarity. The RNAseq data was generated on Illumina NextSeq500, 75 single-end read, high output mode (Illumina, cat no. 200249) The number of reads per gene was counted using Htseq-count (v0.9.1) Results: Transcriptomics of Entamoeba histolytica trophozoites that were adapted to 2 uM Auranofin revealed an upregulation of genes encoding cytoskeletal proteins, dehydrogenases and guanyl-nucleotide exchange factors. Conclusions: Adaptation to Auranofin comes with a fitness cost for E.histolytica that includes a decreased growth rate and virulence and sensitivity to Oxidative stress, Nitrosative stress and to Metronidazole. Overexpression of genes whose products are sensitive to Auranofin-mediated oxidation may represent an important step in the adaptation process to Auranofin and EhTrxR does not seem to be central for this process. Overall design: Entamoeba histolytica mRNA profiles of WT and Auranofin adapted (2uM) trophozoites

研究目的：对适应金诺芬（Auranofin）的溶组织内阿米巴（Entamoeba histolytica）滋养体开展转录组表征。依照美国Sigma-Aldrich公司TRI试剂试剂盒（TRI reagent kit）的说明书，从野生型（WT）对照滋养体与金诺芬适应株（AFAT）中提取总RNA。采用Illumina公司的NEBNext UltraII定向RNA文库制备试剂盒（NEBNext UltraII Directional RNA Library Prep Kit for Illumina，货号E7760），按照制造商方案构建6个RNA测序文库，每个文库使用800ng总RNA。使用磁珠分离模块（NEB，货号E7490）完成mRNA富集。将所有文库以等摩尔浓度混合至单管中。依托Illumina NextSeq500平台，采用75bp单端读长、高产出模式进行测序（Illumina，货号200249）。使用Htseq-count（v0.9.1）统计每个基因的读段数。研究结果：针对适应2μM金诺芬的溶组织内阿米巴滋养体的转录组分析显示，编码细胞骨架蛋白、脱氢酶及鸟苷酸交换因子的基因呈现上调表达。研究结论：溶组织内阿米巴适应金诺芬的过程伴随适合度代价，具体包括生长速率下降、毒力降低，以及对氧化应激、硝化应激和甲硝唑（Metronidazole）的敏感性升高。对金诺芬介导的氧化作用敏感的基因过表达，可能是适应金诺芬过程中的关键步骤，而EhTrxR似乎并非该适应过程的核心因子。实验整体设计：野生型与2μM金诺芬适应的溶组织内阿米巴滋养体的mRNA转录谱分析。