Structure and dynamics of FosA-mediated fosfomycin resistance in Klebsiella pneumoniae and Escherichia coli

Erik H. Klontz, Adam D. Tomich, Sebastian Günther, Justin A. Lemkul, Daniel Deredge, Zach Silverstein, Jo Anna F. Shaw, Christi McElheny, Yohei Doi, Patrick L. Wintrode, Alexander D. MacKerell, Nicolas Sluis-Cremer, Eric J. Sundberg

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Abstract

Fosfomycin exhibits broad-spectrum antibacterial activity and is being reevaluated for the treatment of extensively drug-resistant pathogens. Its activity in Gram-negative organisms, however, can be compromised by expression of FosA, a metal-dependent transferase that catalyzes the conjugation of glutathione to fosfomycin, rendering the antibiotic inactive. In this study, we solved the crystal structures of two of the most clinically relevant FosA enzymes: plasmid-encoded FosA3 from Escherichia coli and chromosomally encoded FosA from Klebsiella pneumoniae (FosAKP). The structure, molecular dynamics, catalytic activity, and fosfomycin resistance of FosA3 and FosAKP were also compared to those of FosA from Pseudomonas aeruginosa (FosAPA), for which prior crystal structures exist. E. coli TOP10 transformants expressing FosA3 and FosAKP conferred significantly greater fosfomycin resistance (MIC, 1,024 g/ml) than those expressing FosAPA (MIC, 16 g/ml), which could be explained in part by the higher catalytic efficiencies of the FosA3 and FosAKP enzymes. Interestingly, these differences in enzyme activity could not be attributed to structural differences at their active sites. Instead, molecular dynamics simulations and hydrogen-deuterium exchange experiments with FosAKP revealed dynamic interconnectivity between its active sites and a loop structure that extends from the active site of each monomer and traverses the dimer interface. This dimer interface loop is longer and more extended in FosAKP and FosA3 than in FosAPA, and kinetic analyses of FosAKP and FosAPA loop-swapped chimeric enzymes highlighted its importance in FosA activity. Collectively, these data yield novel insights into fosfomycin resistance that could be leveraged to develop new strategies to inhibit FosA and potentiate fosfomycin activity.

Original languageEnglish
Article numbere01572-17
JournalAntimicrobial agents and chemotherapy
Volume61
Issue number11
DOIs
Publication statusPublished - 11-2017
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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    Klontz, E. H., Tomich, A. D., Günther, S., Lemkul, J. A., Deredge, D., Silverstein, Z., Shaw, J. A. F., McElheny, C., Doi, Y., Wintrode, P. L., MacKerell, A. D., Sluis-Cremer, N., & Sundberg, E. J. (2017). Structure and dynamics of FosA-mediated fosfomycin resistance in Klebsiella pneumoniae and Escherichia coli. Antimicrobial agents and chemotherapy, 61(11), [e01572-17]. https://doi.org/10.1128/aac.01572-17