Functional insights into ABC-F proteins that mediate antibiotic resistance in Gram-positive bacteria

Members of the ABC-F subfamily of ATP-binding cassette (ABC) proteins mediate resistance to a broad array of clinically-important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which the ABC-F proteins mediate antibiotic resistance is poorly defined, although two hypotheses have been proposed; drug efflux and ribosomal protection. Here, this mechanism of resistance was investigated using a combination of bacteriological and biochemical techniques. Results obtained from the bacteriological assays provided preliminary data in support of ribosomal protection. Subsequently, the heterologous expression and purification of two ABC-F proteins, Vga(A) and Lsa(A), allowed the function of these proteins to be assessed in staphylococcal transcription-translation (T/T) reactions. Addition of Vga(A) and Lsa(A) to T/T assays subject to antibiotic inhibition caused drug specific, dose-dependent, rescue of translation. Several previously described resistance phenotypes attributed to these proteins were successfully recapitulated in T/T assays, corroborating the idea that rescue of translation observed in vitro is representative of the action of these proteins in whole cells. Finally, ribosome binding assays showed Lsa(A) to be capable of displacing antibiotics from staphylococcal ribosomes. Collectively, the experiments described in this thesis provide the first direct evidence to support a mechanism of ARE ABC-F resistance based on ribosomal protection.