Elucidation of role of an acetyltransferase like protein in paromomycin resistance in Leishmania donovaniusing in silicoand in vitroapproaches

AbstractParomomycin, 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 LdATLPwas consistently up-regulated (>2fold) in three distinct paromomycin resistant in comparison with sensitive parasites, although the gene sequence was identical in the two. In silicoanalysis revealed that LdATLPconsisted of conserved GNAT (GCN5-related N-Acetyltransferase) domain which is characteristic of aminoglycoside N-acetyltransferases. Evolutionary relationship among LdATLPof Leishmaniaand 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 LdATLPwith 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 LdATLPwith comparable free energy of binding. In vitrostudies revealed that in the presence of indolicidin, paromomycin resistant parasites exhibited reversion of phenotype into sensitive parasites with marked increase in paromomycin susceptibility, suggesting the role of LdATLPin paromomycin resistance.Communicated by Ramaswamy H. Sarma