Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus

• Published in: The Journal of biological chemistry 2012-08, Vol.287 (36), p.30257-30267
• Main Authors: Koripella, Ravi Kiran, Chen, Yang, Peisker, Kristin, Koh, Cha San, Selmer, Maria, Sanyal, Suparna
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; Antibiotic Resistance; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Crystallography, X-Ray; Drug Resistance, Bacterial; Fusidic Acid - chemistry; Fusidic Acid - pharmacology; GTPase; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - genetics; Guanosine Triphosphate - metabolism; Mutation, Missense; Peptide Elongation Factor G - chemistry; Peptide Elongation Factor G - genetics; Peptide Elongation Factor G - metabolism; Protein Synthesis and Degradation; Ribosomes - chemistry; Ribosomes - genetics; Ribosomes - metabolism; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Transfer - metabolism; Staphylococcus aureus; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics; Translation; Translation Elongation Factors
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M112.378521

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. Background: Fusidic acid-resistant EF-G mutants of Staphylococcus aureus show fitness loss and compensation. Results: Slower translocation and ribosome recycling from restricted conformational change, plus increased tRNA drop-off, cause fitness loss in F88L, which are recovered in F88L/M16I, leading to fitness compensation. Conclusion: Conformational dynamics of EF-G is crucial for function. Significance: This work clarifies how antibiotic-resistant mutations affect in vivo fitness.