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Predicting the emergence of antibiotic resistance by directed evolution and structural analysis


Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 β-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations (500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 Å. G238S widens access to the active site by 2.8 Å whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 Å from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.

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Figure 1: Spectrum of clinical and directed evolution mutation frequencies.
Figure 2: Trends in mutation combinations and order of appearance (other mutations are observed, see Fig 1).
Figure 3: Alternative views of the TEM-52 crystal structure.
Figure 4: Global suppressor mutation M182T found in TEM-52.

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This work was supported in part by the Scripps Research Institute.

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Correspondence to Raymond C. Stevens.

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Orencia, M., Yoon, J., Ness, J. et al. Predicting the emergence of antibiotic resistance by directed evolution and structural analysis. Nat Struct Mol Biol 8, 238–242 (2001).

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