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

Abstract

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.

Figure 1

Sequence alignment of Eis_Msm from M. smegmatis str. MC2 155 and Eis_Mtb from M. tuberculosis H37Rv. The two Eis homologs exhibit 58% sequence identity. Based on structural and mutagenesis studies of Eis_Mtb, the residues proposed to be involved in catalysis, in binding CoA, and in formation of the AG-binding pocket are marked by yellow, orange, and turquoise circles, respectively.(13) Non-conserved residues in the AG-binding pockets of these two proteins are additionally marked by dark blue circles. Conserved residues in the AG-binding pockets of Eis_Msm and Eis_Mtb are marked by green circles.

Figure 2

A. Conversion of NEA into its acetylated products, as monitored by the UV-Vis assay when using Eis_Msm with 1 (black circles) and 10 (white circles) equivalents of AcCoA, respectively. B. A mass spectrum confirming the formation of tri-acetyl-NEA by Eis_Msm. C. Multi-acetylation of NEA by Eis_Msm observed by the TLC assay. Lanes 1–7: a time course displaying the mono-, di-, and tri-acetyl- NEA products of the Eis_Msm reaction. Lanes 8–13: a time course of the NEA reaction with Eis_Mtb. Lanes 14–20: Controls for di- and mono-acetylation of NEA performed with AAC(2′)-Ic, AAC(3)-IV, and AAC(6′) sequentially or individually.

Figure 3

Inhibition of Eis_Msm. IC₅₀ curves for A. chlorhexidine (1), and B. compound 2. The insets show the competitive and mixed inhibition modes of compounds 1 and 2, respectively.

Figure 4

Structural differences between the AG-binding pockets of Eis_Mtb and Eis_Msm. A. The active site and the AG-binding pocket of Eis_Mtb bound to CoA and an acetamide, as seen in the recently reported crystal structure (PDB code: 3R1K (13)). In both panels, CoA is shown in orange, acetamide in red, the conserved AG binding pocket residues in green, and the non-conserved residues in blue. The C-terminal carboxyl group is denoted as C-ter. B. A model of the active site and the AG-binding pocket of Eis_Msm. This model was generated by substituting the non-conserved residues of the AG-binding pocket Eis_Mtb with their Eis_Msm counterparts (Ile268, Trp289, and Gln291 in Eis_Mtb were mutated to Gly266, Ala287, and Ala289 in the Eis_Msm numbering, respectively). As a result of these mutations an APR molecule (in yellow) can be accommodated in the pocket in the orientation appropriate for catalysis. The structure of APR was taken from the high-resolution PDB entry 2OE5.(27)