Murta Silvane M F, Nogueira Fernanda B, Dos Santos Paula F, Campos Fernanda M F, Volpe Caroline, Liarte Daniel B, Nirdé Philippe, Probst Christian M, Krieger Marco A, Goldenberg Samuel, Romanha Alvaro J
Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
Acta Trop. 2008 Jul;107(1):59-65. doi: 10.1016/j.actatropica.2008.04.011. Epub 2008 Apr 18.
Differential gene expression in three pairs of Trypanosoma cruzi populations or clones susceptible or resistant to benznidazole (BZ) was investigated by differential display (DD) and representation of differential expression (RDE). GenBank searches of 14 genes selected by DD showed that four sequences corresponded to different hypothetical proteins and the others were very similar to T. cruzi genes encoding mucin (TcMUC), dihydrolipoamide dehydrogenase (TcLipDH), the hexose transporter (TcHT), or a ribosomal protein. Sequence analysis was performed on 34 clones obtained by RDE; approximately half of these clones encoded 14 different hypothetical proteins and the other half encoded proteins involved with stress response, antioxidant defence, metabolism, transporter proteins, surface proteins, ribosomal proteins and others. The mRNA levels of eight T. cruzi genes obtained by RDE and DD were analysed by northern blotting to confirm the differential expression of these sequences. For six of the eight genes, TcLipDH, TcHT, TcFeSOD-A (iron superoxide dismutase-A), TcHSP70, TcHSP100 (heat shock protein) and Tc52 (thiol-transferase), mRNA levels in the drug-resistant T. cruzi population were at least twice those in the susceptible population. Further analysis of TcHSP70 showed that although the levels of TcHSP70 mRNA were four-fold higher in T. cruzi BZ-resistant population, no corresponding increase was observed in the levels of TcHSP70 protein expression. The results suggest that TcHSP70 is not directly associated with the T. cruzi drug resistance phenotype.
通过差异显示（DD）和差异表达表征（RDE）研究了三对克氏锥虫群体或克隆对苯并硝唑（BZ）敏感或耐药的差异基因表达。对通过DD选择的14个基因进行GenBank搜索，结果显示4个序列对应不同的假定蛋白，其他序列与克氏锥虫编码粘蛋白（TcMUC）、二氢硫辛酰胺脱氢酶（TcLipDH）、己糖转运蛋白（TcHT）或核糖体蛋白的基因非常相似。对通过RDE获得的34个克隆进行了序列分析；这些克隆中约一半编码14种不同的假定蛋白，另一半编码与应激反应、抗氧化防御、代谢、转运蛋白、表面蛋白、核糖体蛋白等相关的蛋白。通过Northern印迹分析了通过RDE和DD获得的8个克氏锥虫基因的mRNA水平，以确认这些序列的差异表达。对于8个基因中的6个，即TcLipDH、TcHT、TcFeSOD-A（铁超氧化物歧化酶-A）、TcHSP70、TcHSP100（热休克蛋白）和Tc52（硫醇转移酶），耐药克氏锥虫群体中的mRNA水平至少是敏感群体中的两倍。对TcHSP70的进一步分析表明，尽管克氏锥虫BZ耐药群体中TcHSP70 mRNA水平高4倍，但未观察到TcHSP70蛋白表达水平相应增加。结果表明，TcHSP70与克氏锥虫耐药表型无直接关联。
