Abstract：
Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.
摘要：
五价抗菌药物是针对不同临床形式的利什曼病的主要治疗方法。流行地区耐药分离株的出现导致治疗失败。解开潜在的抗性机制将有助于改善针对抗性分离株的治疗策略。本研究旨在探讨谷胱甘肽合成酶(GS)的RNA表达水平,亚精胺合成酶(SpS),参与锥硫酮合成的锥硫酮合成酶（TryS）基因，和硫醇依赖性还原酶（TDR）涉及药物减少，在锑敏感和抗性热带利什曼原虫分离株中。我们调查了ACL患者的11种耐锑和11种对锑敏感的热带乳杆菌临床分离株。在小鼠巨噬细胞系J774A.1中确定了amastigotes的药物敏感性。通过定量实时PCR分析前鞭毛形式中的RNA表达水平。结果表明，GS的平均表达显着增加，SpS,与敏感株相比，耐药株中的TrpS基因分别为2.19、1.56和2.33倍。在抗性分离株中TDR的平均表达高1.24倍，这是微不足道的。抑制浓度(IC50)值与基因表达的相关系数最高,属于TryS,GS,SpS,和TDR基因。此外，与敏感菌株相比，耐药菌株的细胞内硫醇含量增加了2.17倍，并且与IC50值正相关。我们的发现表明，锥虫硫生物合成基因的过度表达和硫醇含量的增加可能在热带乳杆菌临床分离株的锑抗性中起关键作用。此外,Tropica临床分离株的锥虫硫酮系统中基因表达的多样性和巯基含量突出了寄生虫种群中锑抗性的表型异质性。
