Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

Abstract

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AAC's). Thus, the modification of aminoglycosides by AAC(2') from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC (3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2'). Interestingly, these effects mirror the biological functions of these enzymes: the AAC (3) used in this study is known to confer aminoglycoside resistance while the AAC(2') is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.

Antimicrobial Susceptibility Testing, Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria

“…The cloning and overexpression of the aminoglycoside acetyltransferase gene aac(2=)-Id of M. smegmatis conferred a 4-to 16-fold increase in the MIC of a range of aminoglycosides, and the disruption of the gene resulted in a drop in the MIC of an equivalent magnitude (4,5). In contrast, the expression of the M. tuberculosis putative aminoglycoside acetyltransferase in M. smegmatis did not increase resistance to this class of agent, despite the demonstration that the enzyme can modify aminoglycosides (24). Regardless of the presence of aminoglycoside acetyltransferase-encoding genes in mycobacteria, the results of several studies indicated that mycobacterial acetyltransferase enzymes do not correlate with susceptibility to these agents (155,419).…”

Glycan Microarrays for Decoding the Glycome

“…In the same year, another cycloaddition, the increasingly utilized “click” reaction, was also employed to array a diverse set of of di, tri-, and tetrasaccharides in microtiter plates (20) ( L ). Although, not broadly used for arraying DNA and peptides, a number of notable publications utilizing click chemistry to array glycans (80, 81), aminoglycosides (82), and glycopolymers (83) have emerged. Moreover, the more recently developed method of microcontact printing has been used to array glycans via both of the above chemistries (81, 83, 84).…”

Studying Modification of Aminoglycoside Antibiotics by Resistance-Causing Enzymes via Microarray

“…Continue to Subheading 3.2, step 6. For the series of aminoglycosides that we have tested in modification assays, all have been functionalized with an azide tag for surface immobilization (34, 36, 39). …”

“…Based on these observations and the development of carbohydrate microarray technology (27–33), our group has developed a microarray approach that monitors aminoglycoside modification by resistance-causing enzymes and the effect of modification on rRNA A-site binding using site-specifically immobilized aminoglycoside substrates (34, 35). These studies, therefore, extend previous investigations that used nonspecifically immobilized aminoglycosides to only probe their binding to RNA (29) or resistance-causing enzymes (27).…”

“…The preparation of the agarose microarray surfaces is critical for these studies, as agarose provides a porous layer that allows for high ligand loading and supports high-density modification of sugars displayed on the array surface (as much as 80% of the total amount of immobilized aminoglycoside) (34, 35). Agarose array surfaces are also quite versatile in the number of chemically reactive handles that can be introduced onto the surface as reactions with amines, alkynes, and azides have all been reported (Fig.…”