Dynamics and quantitative contribution of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] to amikacin resistance

Abstract

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6′)-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6′)-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we combined a fluorescent-based system with CRISPR interference method to modulate and quantify the number of AAC(6′)-Ib per cell in Escherichia coli. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6′)-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6′)-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6′)-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.

Structure-activity relationship of pyrrolidine pentamine derivatives as inhibitors of the aminoglycoside 6′-N-acetyltransferase type Ib

“…1A and Table 1). However, recent work revealed that some bacterial strains produce AAC(6′)-Ib in quantities far exceeding those needed for clinical resistance [16, 31]. This finding highlights the necessity for an inhibitor with enough potency to effectively combat a range of pathogenic bacteria, even in environments with high enzyme concentrations.…”