Aminoglycoside multiacetylating activity of the enhanced intracellular survival protein from Mycobacterium smegmatis and its inhibition

The enhanced intracellular survival (Eis) protein improves the survival of Mycobacterium smegmatis (Msm) 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 Mycobacterium tuberculosis (Mtb) clinical isolates. We recently demonstrated that Eis from Mtb (Eis-Mtb) 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-Msm, investigated its inhibition, and compared these functions to those of Eis-Mtb. Even though for several AGs the multiacetylation properties of Eis-Msm and Eis-Mtb are similar, there are three major differences. (i) Eis-Msm diacetylates apramycin, a conformationally constrained AG, which Eis-Mtb cannot modify. (ii) Eis-Msm triacetylates paromomycin, which can be only diacetylated by Eis-Mtb. (iii) Eis-Msm only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis-Mtb. 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-Msm is more open than that of Eis-Mtb, it accommodates apramycin for acetylation in Eis-Msm, but not in Eis-Mtb. We also demonstrate that inhibitors of Eis-Mtb that we recently discovered can inhibit Eis-Msm activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis-Mtb inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation (Chemical Equation Presented).