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 the 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.Communicated by Ramaswamy H. Sarma.