Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Sciences


Microbiology, Immunology, and Biochemistry

Research Advisor

Richard E. Lee, PhD


Hans Haecker, MD, PhD David R. Nelson, PhD P. David Rogers, PharmD, PhD, FCCP Jason W. Rosch, PhD


Aminoglycoside modifying enzymes, Antibacterial Drug Discovery; Antimicrobial Resistance, Gram- negative bacteria, Spectinomycin


Spectinomycin (SPC) is a broad-spectrum aminocyclitol antibiotic. Its use in agriculture has led to widespread resistance in enteric bacteria, necessitating the development of more effective analogs. Aminomethyl spectinomycins (amSPC) are modified spectinomycins with increased potency against many bacterial species. These species include Legionella pneumophila, which harbors a chromosomally encoded aminoglycoside modifying enzyme (AME). In this study, we follow up on this observation and examine the extent to which the amSPCs are substrates for AMEs through adenylation (ANTs) and phosphorylation (APH). APH(9)-Ia and ANT(3")(9) were expressed in E. coli BL21(DE3) and purified using the Ni-affinity chromatography. The ability of AMEs to modify and inactivate amSPCs has been examined by two unique biochemical assays, including an agar-based enzyme assay. Binding of APH (9)-Ia and ANT (3")(9) to spectinomycin and amSPCs has been studied using Thermal Denaturation assay and MicroScale Thermophoresis (MST).

The microbiological role of these enzymes has been examined by minimum inhibitory concentration (MIC) shifts using an arabinose inducible expression of APH (9)-Ia and ANT (3")(9) in E.coli K12 and JW ΔtolC strains. Our agar-based enzyme assay shows the inactivation of spectinomycin by APH(9)-Ia. Phosphorylated spectinomycin and adenylated spectinomycin products upon incubation with APH(9)-Ia and ANT(3",9), respectively, have been identified using MALDI-MS. APH(9)-Ia induction studies in E. coli tolC knock-out strains reveal a MIC increase against spectinomycin in the presence of 2% arabinose compared to no shift with amSPCs. ANT (3")(9) showed an increase in MIC against spectinomycin as well as amSPCs. In conclusion, amSPCs are not inactivated by APH (9)-Ia in vivo but are inactivated by ANT (3")(9). Most Gram-negative bacteria isolated in clinics possess one or more AMEs. By overcoming modification by AMEs, amSPCs can be a valuable tool in overcoming resistance in Gram-negative bacterial infections. We also conducted a high throughput screen of a polar small molecule library against two multi-drug resistant clinical isolates of Escherichia coli that encode aminoglycoside modifying enzyme for small molecule potentiators of amSPCs to yield 12 possible potentiating molecules that have been confirmed by dose-response analysis.

Future work as a continuation of this project will involve further analysis of any existing synergy between the potentiating molecules and amSPCs and target validation of these potentiators.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.




2021-027-Das-DOA.pdf (171 kB)
Declaration of Authorship