摘要

帕龙霉素是一种氨基糖苷类抗生素，在印度对VL（内脏利什曼病）有效。诸如氨基糖苷乙酰基转移酶之类的酶对氨基糖苷抗生素的修饰是细菌系统中对抗生素产生抗性的主要机制。在本研究中，我们鉴定并鉴定了LdATLP（一种类似乙酰基转移酶的蛋白），并阐明了其在利什曼原虫的帕龙霉素抗性中的作用。与敏感的寄生虫相比，编码LdATLP的基因在三种不同的帕龙霉素耐药性中始终被上调（> 2倍），尽管两者的基因序列相同。在计算机分析中发现，LdATLP由保守的GNAT（GCN5相关的N-乙酰基转移酶）结构域组成，其是氨基糖苷N-乙酰基转移酶的特征。通过系统进化分析，建立了利什曼原虫LdATLP与细菌氨基糖苷乙酰基转移酶之间的进化关系。由ab-initio建模预测的LdATLP的3D结构构成6个α-螺旋和6个β-折叠。预测了GNAT域的一些残基，例如R175，R177，E196，R197，V198，V200，K202，R205，C206，D208，G210，R211，R215，A234，S237，S238，K239，D240，F241和Y242出现在活动现场。 LdATLP与巴龙霉素或吲哚icidin（氨基糖苷修饰酶的广谱抑制剂）分子对接，然后对接复合物的分子动力学模拟表明，帕龙霉素和吲哚美菌素均以相当的结合自由能与LdATLP结合。体外研究表明，在吲哚美定存在的情况下，耐帕龙霉素的寄生虫表现出表型转化为敏感的寄生虫，帕龙霉素的敏感性显着增加，表明LdATLP在帕龙霉素耐药性中的作用。

Paromomycin, an aminoglycoside antibiotic, is an effective treatment for VL (visceral leishmaniasis) in India. The modification of aminoglycoside antibiotics by enzymes such as aminoglycoside acetyltransferases is the predominant mechanism of resistance to antibiotics in bacterial system. In the present study, we identified and characterized LdATLP (an acetyltransferase like protein) and elucidated its role in paromomycin resistance in Leishmania donovani. Gene encoding LdATLP was consistently up-regulated (>2fold) in three distinct paromomycin resistant in comparison with sensitive parasites, although the gene sequence was identical in the two. In silico analysis revealed that LdATLP consisted of conserved GNAT (GCN5-related N-Acetyltransferase) domain which is characteristic of aminoglycoside N-acetyltransferases. Evolutionary relationship among LdATLP of Leishmania and aminoglycoside acetyltransferases of bacteria was established by phylogenetic analysis. The 3D structure of LdATLP, predicted by ab-initio modeling, constituted 6 α-helices and 6 β-sheets. A few residues, such as R175, R177, E196, R197, V198, V200, K202, R205, C206, D208, G210, R211, R215, A234, S237, S238, K239, D240, F241 and Y242 of GNAT domain were predicted to be present at active site. Molecular docking of LdATLP with paromomycin or indolicidin (broad spectrum inhibitor of aminoglycoside modifying enzymes), followed by molecular dynamics simulation of docked complex suggested that both paromomycin and indolicidin bind to LdATLP with comparable free energy of binding. In vitro studies revealed that in presence of indolicidin, paromomycin resistant parasites exhibited reversion of phenotype into sensitive parasites with marked increase in paromomycin susceptibility, suggesting the role of LdATLP in paromomycin resistance.

https://www.ncbi.nlm.nih.gov/pubmed/31625467