Structural insights into ribosomes from pathogenic bacteria and their complexes with antibiotics and antibiotic-resistance ABC-F proteins

Ribosome-targeting antibiotics are commonly used to treat infections caused by pathogenic bacteria, yet, concerningly, resistance to these drugs often develops following their introduction to the clinic. To effectively combat this threat, our understanding of the mechanisms by which antibiotics work and by which bacteria acquire resistance must be improved. To this end, this thesis presents an analysis of the cryo-electron microscope (cryo-EM) structures of ribosomes from pathogenic bacteria in complex with either an antibiotic or an ATP-binding cassette (ABC)-F antibiotic resistance protein. Structures of the ribosome from Acinetobacter baumannii in complex with clinically relevant antibiotics amikacin or tigecycline reveal species-specific ribosomal features, particularly around the exit of the nascent peptide exit tunnel (NPET) and around the edge of the subunit interface. Amikacin and tigecycline interact with the A. baumannii ribosome at known aminoglycoside and tetracycline small subunit binding sites, respectively; however, tigecycline unexpectedly also binds at a secondary site within the central protuberance of the large subunit. The structure of the ribosome from Staphylococcus aureus in complex with the antibiotic resistance ABC-F protein Sal(B) reveals that this protein binds the E site of the ribosome, with its interdomain linker protruding towards the peptidyl transferase centre (PTC), where it likely displaces pleuromutilin, lincosamide and streptogramin A antibiotics by a purely allosteric mechanism. Mutagenesis experiments and sequence comparison with Sal variants that mediate different resistance phenotypes show that tyrosine-264 of Sal(B) plays an important role in resistance, but that other residues must also contribute. Overall, the work outlined in this thesis contributes to our molecular understanding of antibiotic action and resistance, and makes steps towards the structure-based rational design or modification of drugs that have improved activity against pathogens such as A. baumannii or S. aureus, or that have the ability to circumvent or inhibit the ABC-F target protection resistance mechanism.