Aminoglycoside Multiacetylating Activity of the Enhanced Intracellular Survival Protein from <i>Mycobacterium smegmatis</i> and Its Inhibition

Abstract

The enhanced intracellular survival (Eis) protein improves the survival of <i>Mycobacterium smegmatis</i> (<i>Msm</i>) in macrophages and functions as the acetyltransferase responsible for kanamycin A resistance, a hallmark of extensively drug-resistant (XDR) tuberculosis, in a large number of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) clinical isolates. We recently demonstrated that Eis from <i>Mtb</i> (Eis_<i>Mtb</i>) efficiently multiacetylates a variety of aminoglycoside (AG) antibiotics. Here, to gain insight into the origin of substrate selectivity of AG multiacetylation by Eis, we analyzed AG acetylation by Eis_<i>Msm</i>, investigated its inhibition, and compared these functions to those of Eis_<i>Mtb</i>. Even though for several AGs the multiacetylation properties of Eis_<i>Msm</i> and Eis_<i>Mtb</i> are similar, there are three major differences. (i) Eis_<i>Msm</i> diacetylates apramycin, a conformationally constrained AG, which Eis_<i>Mtb</i> cannot modify. (ii) Eis_<i>Msm</i> triacetylates paromomycin, which can be only diacetylated by Eis_<i>Mtb</i>. (iii) Eis_<i>Msm</i> only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis_<i>Mtb</i>. Several nonconserved amino acid residues lining the AG-binding pocket of Eis are likely responsible for these differences between the two Eis homologues. Specifically, we propose that because the AG-binding pocket of Eis_<i>Msm</i> is more open than that of Eis_<i>Mtb</i>, it accommodates apramycin for acetylation in Eis_<i>Msm</i>, but not in Eis_<i>Mtb</i>. We also demonstrate that inhibitors of Eis_<i>Mtb</i> that we recently discovered can inhibit Eis_<i>Msm</i> activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis_<i>Mtb</i> inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation