title: Mechanism of elongation factor-G-mediated fusidic acid resistance and fitness compensation in Staphylococcus aureus.
abstract: Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.
authors: Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden.
authors: Ravi Kiran
authors: RK
authors: Koripella
authors: Yang
authors: Y
authors: Chen
authors: Kristin
authors: K
authors: Peisker
authors: Cha San
authors: CS
authors: Koh
authors: Maria
authors: M
authors: Selmer
authors: Suparna
authors: S
authors: Sanyal
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D005672:Fusidic Acid / Q000737:chemistry* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C492444:FusA protein, Staphylococcus aureus; D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D012343:RNA, Transfer