title: Biophysical principles predict fitness landscapes of drug resistance.
abstract: Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molecular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Further, we found that a controlled modulation of the GroEL/ES chaperonins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This unveils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolutionary outcome. These results provide a comprehensive quantitative genotype-phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138;
authors.forename: João V
authors.identifier: http://orcid.org/0000-0002-5605-656X
authors.initials: JV
authors.lastname: Rodrigues
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel;
authors.forename: Shimon
authors.initials: S
authors.lastname: Bershtein
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge MA 02138.
authors.forename: Anna
authors.initials: A
authors.lastname: Li
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge MA 02138.
authors.forename: Elena R
authors.initials: ER
authors.lastname: Lozovsky
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge MA 02138 dhartl@oeb.harvard.edu shakhnovich@chemistry.harvard.edu.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Hartl
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; dhartl@oeb.harvard.edu shakhnovich@chemistry.harvard.edu.
authors.forename: Eugene I
authors.initials: EI
authors.lastname: Shakhnovich
mesh_terms: D000595:Amino Acid Sequence; D001703:Biophysics / Q000379:methods; D023502:Chlamydia muridarum / Q000235:genetics; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics; D004843:Epistasis, Genetic; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008087:Listeria / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D029968:Escherichia coli Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase
keywords: DHFR; drug resistance; fitness landscapes; molten globule; protein stability