抗锑和敏感的巴西利什曼原虫及婴儿利什曼原虫系中胞质硫氧还蛋白过氧化物酶的功能分析
Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and -susceptible Leishmania braziliensis and Leishmania infantum lines.
作者信息
Andrade Juvana M, Murta Silvane M F
机构信息
Centro de Pesquisas René Rachou/FIOCRUZ, Avenida Augusto de Lima 1715, Belo Horizonte 30190-002, MG, Brazil.
出版信息
Parasit Vectors. 2014 Aug 29;7:406. doi: 10.1186/1756-3305-7-406.
BACKGROUND
Tryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxidative stress.
METHODS
In this study, the gene encoding cytosolic TXNPx (cTXNPx) was characterized in lines of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum that are susceptible and resistant to potassium antimony tartrate (Sb(III)). We investigated the levels of mRNA and genomic organization of the cTXNPx gene. In addition, we transfected the Leishmania lines with the cTXNPx gene and analysed the susceptibility of transfected parasites to Sb(III) and to hydrogen peroxide (H2O2).
RESULTS
Northern blot and real-time reverse transcriptase polymerase chain reaction analyses revealed that the level of TXNPx mRNA was approximately 2.5-fold higher in the Sb(III)-resistant L. braziliensis line than in the parental line. In contrast, no significant difference in cTXNPx mRNA levels between the L. infantum lines was observed. Southern blot analyses revealed that the cTXNPx gene is not amplified in the genome of the Sb(III)-resistant Leishmania lines analysed. Functional analysis of cTXNPx was performed to determine whether overexpression of the enzyme in L. braziliensis and L. infantum lines would change their susceptibility to Sb(III). Western blotting analysis showed that the level of cTXNPx was 2 to 4-fold higher in transfected clones compared to non-transfected cells. Antimony susceptibility test (EC50 assay) revealed that L. braziliensis lines overexpressing cTXNPx had a 2-fold increase in resistance to Sb(III) when compared to the untransfected parental line. In addition, these clones are more tolerant to exogenous H2O2 than the untransfected parental line. In contrast, no difference in Sb(III) susceptibility and a moderate index of resistance to H2O2 was observed in L. infantum clones overexpressing cTXNPx.
CONCLUSION
Our functional analysis revealed that cTXNPx is involved in the antimony-resistance phenotype in L. braziliensis.
背景
锥虫硫氧还蛋白过氧化物酶（TXNPx）作为一种抗氧化剂，通过将过氧化氢代谢为水分子参与抗氧化应激防御。报告表明，耐药寄生虫可能会提高TXNPx和其他酶的水平，从而保护它们免受氧化应激。
方法
在本研究中，对巴西利什曼原虫（维氏亚属）和婴儿利什曼原虫（利什曼亚属）中编码胞质TXNPx（cTXNPx）的基因进行了表征，这些虫株对酒石酸锑钾（Sb(III)）敏感或耐药。我们研究了cTXNPx基因的mRNA水平和基因组结构。此外，我们用cTXNPx基因转染利什曼原虫株，并分析转染后的寄生虫对Sb(III)和过氧化氢（H2O2）的敏感性。
结果
Northern印迹和实时逆转录聚合酶链反应分析显示，耐Sb(III)的巴西利什曼原虫株中TXNPx mRNA水平比亲代株高约2.5倍。相比之下，婴儿利什曼原虫株之间的cTXNPx mRNA水平未观察到显著差异。Southern印迹分析显示，在所分析的耐Sb(III)利什曼原虫株的基因组中，cTXNPx基因未扩增。对cTXNPx进行功能分析，以确定该酶在巴西利什曼原虫和婴儿利什曼原虫株中的过表达是否会改变它们对Sb(III)的敏感性。蛋白质印迹分析表明，与未转染细胞相比，转染克隆中cTXNPx的水平高2至4倍。锑敏感性试验（EC50测定）显示，过表达cTXNPx的巴西利什曼原虫株与未转染的亲代株相比，对Sb(III)的抗性增加了2倍。此外，这些克隆比未转染的亲代株对外源H2O2更耐受。相比之下，过表达cTXNPx的婴儿利什曼原虫克隆在Sb(III)敏感性方面没有差异，对H2O2的抗性指数中等。
结论
我们的功能分析表明，cTXNPx参与了巴西利什曼原虫的抗锑表型。
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