Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens

Krucinska, Jolanta; Lombardo, Michael N.; Erlandsen, Heidi; Estrada, Alexavier; Si, Debjani; Viswanathan, Kishore; Wright, Dennis L.

doi:10.1038/s42003-022-03384-y

Title: Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens

Journal Article · 13 May 2022 · Communications Biology

DOI: https://doi.org/10.1038/s42003-022-03384-y · OSTI ID:2471731

^[1]; Lombardo, Michael N. ^[1];

^[1];

^[1]; Si, Debjani ^[1]; Viswanathan, Kishore ^[1];

^[1]

University of Connecticut, Storrs, CT (United States)

Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL); Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-76SF00515; SC0012704

OSTI ID:: 2471731

Journal Information:: Communications Biology, Journal Name: Communications Biology Journal Issue: 1 Vol. 5; ISSN 2399-3642

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

References (71)

Are the Catalytic Properties of Enzymes from Piezophilic Organisms Pressure Adapted? Hay, Sam; Evans, Rhiannon M.; Levy, Colin ChemBioChem, Vol. 10, Issue 14 https://doi.org/10.1002/cbic.200900367	journal	September 2009
Effect of pH on Hydride Transfer by Escherichia coli Dihydrofolate Reductase Loveridge, E. Joel; Allemann, Rudolf K. ChemBioChem, Vol. 12, Issue 8 https://doi.org/10.1002/cbic.201000794	journal	April 2011
Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim Heaslet, Holly; Harris, Melissa; Fahnoe, Kelly Proteins: Structure, Function, and Bioinformatics, Vol. 76, Issue 3 https://doi.org/10.1002/prot.22383	journal	August 2009
Structural genomics for drug design against the pathogen Coxiella burnetii : Drug Design Against Franklin, Matthew C.; Cheung, Jonah; Rudolph, Michael J. Proteins: Structure, Function, and Bioinformatics, Vol. 83, Issue 12 https://doi.org/10.1002/prot.24841	journal	October 2015
Structure and energetics of ligand binding to proteins:Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system Dauber-Osguthorpe, Pnina; Roberts, Victoria A.; Osguthorpe, David J. Proteins: Structure, Function, and Genetics, Vol. 4, Issue 1 https://doi.org/10.1002/prot.340040106	journal	January 1988
MICs of ciprofloxacin and trimethoprim for Escherichia coli: Influence of pH, inoculum size and various body fluids Aagaard, J.; Madsen, P. O.; Rhodes, P. Infection, Vol. 19, Issue S3 https://doi.org/10.1007/BF01643691	journal	May 1991
Reduced Susceptibility of Moritella profunda Dihydrofolate Reductase to Trimethoprim is Not Due to Glutamate 28 Loveridge, E. Joel; Dawson, William M.; Evans, Rhiannon M. The Protein Journal, Vol. 30, Issue 8 https://doi.org/10.1007/s10930-011-9361-x	journal	October 2011
Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction Yung-Chi, Cheng; Prusoff, William H. Biochemical Pharmacology, Vol. 22, Issue 23 https://doi.org/10.1016/0006-2952(73)90196-2	journal	December 1973
Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase Champness, J. N.; Stammers, D. K.; Beddell, C. R. FEBS Letters, Vol. 199, Issue 1 https://doi.org/10.1016/0014-5793(86)81224-8	journal	April 1986
Thermodynamic study of dihydrofolate reductase inhibitor selectivity Sasso, Sophie P.; Gilli, Robert M.; Sari, Jean Claude Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol. 1207, Issue 1 https://doi.org/10.1016/0167-4838(94)90053-1	journal	July 1994
Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate. Bolin, J. T.; Filman, D. J.; Matthews, D. A. Journal of Biological Chemistry, Vol. 257, Issue 22 https://doi.org/10.1016/S0021-9258(18)33497-5	journal	November 1982
Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim. Matthews, D. A.; Bolin, J. T.; Burridge, J. M. Journal of Biological Chemistry, Vol. 260, Issue 1 https://doi.org/10.1016/S0021-9258(18)89743-5	journal	January 1985
Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH. Appleman, J. R.; Howell, E. E.; Kraut, J. Journal of Biological Chemistry, Vol. 265, Issue 10 https://doi.org/10.1016/S0021-9258(19)39400-1	journal	April 1990
Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase. Appleman, J. R.; Prendergast, N.; Delcamp, T. J. Journal of Biological Chemistry, Vol. 263, Issue 21 https://doi.org/10.1016/S0021-9258(19)81516-8	journal	July 1988
MRSA Isolates from United States Hospitals Carry dfrG and dfrK Resistance Genes and Succumb to Propargyl-Linked Antifolates Reeve, Stephanie M.; Scocchera, Eric W.; G-Dayanadan, Narendran Cell Chemical Biology, Vol. 23, Issue 12 https://doi.org/10.1016/j.chembiol.2016.11.007	journal	December 2016
New β-Lactamase Inhibitors in the Clinic Papp-Wallace, Krisztina M.; Bonomo, Robert A. Infectious Disease Clinics of North America, Vol. 30, Issue 2 https://doi.org/10.1016/j.idc.2016.02.007	journal	June 2016
Allosteric Communication in Dihydrofolate Reductase: Signaling Network and Pathways for Closed to Occluded Transition and Back Chen, Jie; Dima, Ruxandra I.; Thirumalai, D. Journal of Molecular Biology, Vol. 374, Issue 1 https://doi.org/10.1016/j.jmb.2007.08.047	journal	November 2007
Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternate Conformation of NADPH That May Be Linked to Trimethoprim Resistance Frey, Kathleen M.; Liu, Jieying; Lombardo, Michael N. Journal of Molecular Biology, Vol. 387, Issue 5 https://doi.org/10.1016/j.jmb.2009.02.045	journal	April 2009
Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State Mauldin, Randall V.; Carroll, Mary J.; Lee, Andrew L. Structure, Vol. 17, Issue 3 https://doi.org/10.1016/j.str.2009.01.005	journal	March 2009
Enterobacter meningitis: organism susceptibilities, antimicrobial therapy and related outcomes Foster, David R.; Rhoney, Denise H. Surgical Neurology, Vol. 63, Issue 6 https://doi.org/10.1016/j.surneu.2004.06.018	journal	June 2005
Charged Propargyl-Linked Antifolates Reveal Mechanisms of Antifolate Resistance and Inhibit Trimethoprim-Resistant MRSA Strains Possessing Clinically Relevant Mutations Reeve, Stephanie M.; Scocchera, Eric; Ferreira, Jacob J. Journal of Medicinal Chemistry, Vol. 59, Issue 13 https://doi.org/10.1021/acs.jmedchem.6b00688	journal	June 2016
Direct Substitution of Arylalkynyl Carbinols Provides Access to Diverse Terminal Acetylene Building Blocks G-Dayanandan, Narendran; Scocchera, Eric W.; Keshipeddy, Santosh Organic Letters, Vol. 19, Issue 1 https://doi.org/10.1021/acs.orglett.6b03438	journal	December 2016
Crystal Structures of Trimethoprim-Resistant DfrA1 Rationalize Potent Inhibition by Propargyl-Linked Antifolates Lombardo, Michael N.; G-Dayanandan, Narendran; Wright, Dennis L. ACS Infectious Diseases, Vol. 2, Issue 2 https://doi.org/10.1021/acsinfecdis.5b00129	journal	December 2015
Toward Broad Spectrum Dihydrofolate Reductase Inhibitors Targeting Trimethoprim Resistant Enzymes Identified in Clinical Isolates of Methicillin Resistant Staphylococcus aureus Reeve, Stephanie M.; Si, Debjani; Krucinska, Jolanta ACS Infectious Diseases, Vol. 5, Issue 11 https://doi.org/10.1021/acsinfecdis.9b00222	journal	September 2019
Charged Nonclassical Antifolates with Activity Against Gram-Positive and Gram-Negative Pathogens Scocchera, Eric; Reeve, Stephanie M.; Keshipeddy, Santosh ACS Medicinal Chemistry Letters, Vol. 7, Issue 7 https://doi.org/10.1021/acsmedchemlett.6b00120	journal	May 2016
Dynamics of the Dihydrofolate Reductase-Folate Complex: Catalytic Sites and Regions Known To Undergo Conformational Change Exhibit Diverse Dynamical Features Epstein, David M.; Benkovic, Stephen J.; Wright, Peter E. Biochemistry, Vol. 34, Issue 35 https://doi.org/10.1021/bi00035a009	journal	September 1995
Multistate Equilibrium Unfolding of Escherichia coli Dihydrofolate Reductase: Thermodynamic and Spectroscopic Description of the Native, Intermediate, and Unfolded Ensembles Ionescu, Roxana M.; Smith, Virginia F.; O'Neil, John C. Biochemistry, Vol. 39, Issue 31 https://doi.org/10.1021/bi000511y	journal	July 2000
Complementary perturbation of the kinetic mechanism and catalytic effectiveness of dihydrofolate reductase by side-chain interchange Wagner, Carston R.; Thillet, Joelle; Benkovic, Stephen J. Biochemistry, Vol. 31, Issue 34 https://doi.org/10.1021/bi00149a013	journal	September 1992
Structure and function of alternative proton-relay mutants of dihydrofolate reductase David, Cynthia L.; Howell, Elizabeth E.; Farnum, Martin F. Biochemistry, Vol. 31, Issue 40 https://doi.org/10.1021/bi00155a038	journal	October 1992
Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis Falzone, Christopher J.; Wright, Peter E.; Benkovic, Stephen J. Biochemistry, Vol. 33, Issue 2 https://doi.org/10.1021/bi00168a007	journal	January 1994
Determination by Raman Spectroscopy of the pKa of N5 of Dihydrofolate Bound to Dihydrofolate Reductase: Mechanistic Implications Chen, Y. -Q.; Kraut, Joseph; Blakley, Raymond L. Biochemistry, Vol. 33, Issue 23 https://doi.org/10.1021/bi00189a001	journal	June 1994
Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding Bystroff, Christopher; Kraut, Joseph Biochemistry, Vol. 30, Issue 8 https://doi.org/10.1021/bi00222a028	journal	February 1991
Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase Maharaj, Geeta; Selinsky, Barry S.; Appleman, James R. Biochemistry, Vol. 29, Issue 19 https://doi.org/10.1021/bi00471a008	journal	May 1990
Backbone Dynamics in Dihydrofolate Reductase Complexes: Role of Loop Flexibility in the Catalytic Mechanism ^† Osborne, Michael J.; Schnell, Jason; Benkovic, Stephen J. Biochemistry, Vol. 40, Issue 33 https://doi.org/10.1021/bi010621k	journal	August 2001
Conformational Changes in the Active Site Loops of Dihydrofolate Reductase during the Catalytic Cycle ^† Venkitakrishnan, Rani P.; Zaborowski, Eduardo; McElheny, Dan Biochemistry, Vol. 43, Issue 51 https://doi.org/10.1021/bi048119y	journal	December 2004
A Distal Mutation Perturbs Dynamic Amino Acid Networks in Dihydrofolate Reductase Boehr, David D.; Schnell, Jason R.; McElheny, Dan Biochemistry, Vol. 52, Issue 27 https://doi.org/10.1021/bi400563c	journal	June 2013
Elucidating Features That Drive the Design of Selective Antifolates Using Crystal Structures of Human Dihydrofolate Reductase Lamb, Kristen M.; G-Dayanandan, Narendran; Wright, Dennis L. Biochemistry, Vol. 52, Issue 41 https://doi.org/10.1021/bi400852h	journal	October 2013
Loop and Subdomain Movements in the Mechanism of Escherichia coli Dihydrofolate Reductase: Crystallographic Evidence ^† ^, ^‡ Sawaya, Michael R.; Kraut, Joseph Biochemistry, Vol. 36, Issue 3 https://doi.org/10.1021/bi962337c	journal	January 1997
Nonracemic Antifolates Stereoselectively Recruit Alternate Cofactors and Overcome Resistance in S. aureus Keshipeddy, Santosh; Reeve, Stephanie M.; Anderson, Amy C. Journal of the American Chemical Society, Vol. 137, Issue 28 https://doi.org/10.1021/jacs.5b01442	journal	July 2015
Antibacterial drug discovery in the resistance era Brown, Eric D.; Wright, Gerard D. Nature, Vol. 529, Issue 7586 https://doi.org/10.1038/nature17042	journal	January 2016
Integrating macromolecular X-ray diffraction data with the graphical user interface iMosflm Powell, Harold R.; Battye, T. Geoff G.; Kontogiannis, Luke Nature Protocols, Vol. 12, Issue 7 https://doi.org/10.1038/nprot.2017.037	journal	June 2017
A trimethoprim derivative impedes antibiotic resistance evolution Manna, Madhu Sudan; Tamer, Yusuf Talha; Gaszek, Ilona Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-23191-z	journal	May 2021
Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria Bennett, P. M. British Journal of Pharmacology, Vol. 153, Issue S1 https://doi.org/10.1038/sj.bjp.0707607	journal	March 2008
Increased substrate affinity in the Escherichia coli L28R dihydrofolate reductase mutant causes trimethoprim resistance Abdizadeh, Haleh; Tamer, Yusuf Talha; Acar, Omer Physical Chemistry Chemical Physics, Vol. 19, Issue 18 https://doi.org/10.1039/C7CP01458A	journal	January 2017
Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography Wan, Qun; Bennett, Brad C.; Wilson, Mark A. Proceedings of the National Academy of Sciences, Vol. 111, Issue 51 https://doi.org/10.1073/pnas.1415856111	journal	December 2014
Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100 Liu, C. Tony; Francis, Kevin; Layfield, Joshua P. Proceedings of the National Academy of Sciences, Vol. 111, Issue 51 https://doi.org/10.1073/pnas.1415940111	journal	December 2014
Basis of Selectivity of Antibacterial Diaminopyrimidines Baccanari, D. P.; Kuyper, L. F. Journal of Chemotherapy, Vol. 5, Issue 6 https://doi.org/10.1080/1120009X.1993.11741086	journal	December 1993
Iclaprim: a differentiated option for the treatment of skin and skin structure infections Noviello, Stephanie; Huang, David B.; Corey, G. Ralph Expert Review of Anti-infective Therapy, Vol. 16, Issue 11 https://doi.org/10.1080/14787210.2018.1536545	journal	October 2018
Resistance to Trimethoprim-Sulfamethoxazole Eliopoulos, G. M.; Huovinen, P. Clinical Infectious Diseases, Vol. 32, Issue 11 https://doi.org/10.1086/320532	journal	June 2001
Global Spread of Mobile Antimicrobial Drug Resistance Determinants in Human and AnimalEscherichia coliandSalmonellaStrains Causing Community‐Acquired Infections Ajiboye, Remi M.; Solberg, Owen D.; Lee, Bryan M. Clinical Infectious Diseases, Vol. 49, Issue 3 https://doi.org/10.1086/600301	journal	August 2009
High-Order Epistasis in Catalytic Power of Dihydrofolate Reductase Gives Rise to a Rugged Fitness Landscape in the Presence of Trimethoprim Selection Tamer, Yusuf Talha; Gaszek, Ilona K.; Abdizadeh, Haleh Molecular Biology and Evolution, Vol. 36, Issue 7 https://doi.org/10.1093/molbev/msz086	journal	April 2019
Prevalence of trimethoprim resistance genes in Escherichia coli isolates of human and animal origin in Lithuania Šeputienė, Vaida; Povilonis, Justas; Ružauskas, Modestas Journal of Medical Microbiology, Vol. 59, Issue 3 https://doi.org/10.1099/jmm.0.015008-0	journal	March 2010
Exploration into the origins and mobilization of di-hydrofolate reductase genes and the emergence of clinical resistance to trimethoprim Sánchez-Osuna, Miquel; Cortés, Pilar; Llagostera, Montserrat Microbial Genomics, Vol. 6, Issue 11 https://doi.org/10.1099/mgen.0.000440	journal	November 2020
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
Automated search-model discovery and preparation for structure solution by molecular replacement Keegan, Ronan M.; Winn, Martyn D. Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 4 https://doi.org/10.1107/S0907444907002661	journal	March 2007
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
REFMAC 5 for the refinement of macromolecular crystal structures Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444911001314	journal	March 2011
CCP 4 i 2: the new graphical user interface to the CCP 4 program suite Potterton, Liz; Agirre, Jon; Ballard, Charles Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 2 https://doi.org/10.1107/S2059798317016035	journal	February 2018
Role of water in the catalytic cycle of E. coli dihydrofolate reductase Shrimpton, Paul; Allemann, Rudolf K. Protein Science, Vol. 11, Issue 6 https://doi.org/10.1110/ps.5060102	journal	June 2002
Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria Grape, M.; Farra, A.; Kronvall, G. Clinical Microbiology and Infection, Vol. 11, Issue 3 https://doi.org/10.1111/j.1469-0691.2004.01059.x	journal	March 2005
The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis Boehr, D. D.; McElheny, D.; Dyson, H. J. Science, Vol. 313, Issue 5793, p. 1638-1642 https://doi.org/10.1126/science.1130258	journal	September 2006
Functional role of aspartic acid-27 in dihydrofolate reductase revealed by mutagenesis Howell, E.; Villafranca, J.; Warren, M. Science, Vol. 231, Issue 4742 https://doi.org/10.1126/science.3511529	journal	March 1986
Crystal Structures of Klebsiella pneumoniae Dihydrofolate Reductase Bound to Propargyl-Linked Antifolates Reveal Features for Potency and Selectivity Lamb, Kristen M.; Lombardo, Michael N.; Alverson, Jeremy Antimicrobial Agents and Chemotherapy, Vol. 58, Issue 12 https://doi.org/10.1128/AAC.03555-14	journal	December 2014
Trimethoprim and sulfonamide resistance Huovinen, P.; Sundstrom, L.; Swedberg, G. Antimicrobial Agents and Chemotherapy, Vol. 39, Issue 2 https://doi.org/10.1128/AAC.39.2.279	journal	February 1995
Structure, Dynamics, and Catalytic Function of Dihydrofolate Reductase Schnell, Jason R.; Dyson, H. Jane; Wright, Peter E. Annual Review of Biophysics and Biomolecular Structure, Vol. 33, Issue 1 https://doi.org/10.1146/annurev.biophys.33.110502.133613	journal	June 2004
Molecular Characterisation of Trimethoprim Resistance in Escherichia coli and Klebsiella pneumoniae during a Two Year Intervention on Trimethoprim Use Brolund, Alma; Sundqvist, Martin; Kahlmeter, Gunnar PLoS ONE, Vol. 5, Issue 2 https://doi.org/10.1371/journal.pone.0009233	journal	February 2010
Toward New Therapeutics for Skin and Soft Tissue Infections: Propargyl-Linked Antifolates Are Potent Inhibitors of MRSA and Streptococcus pyogenes Viswanathan, Kishore; Frey, Kathleen M.; Scocchera, Eric W. PLoS ONE, Vol. 7, Issue 2 https://doi.org/10.1371/journal.pone.0029434	journal	February 2012
Trimethoprim-Sulfamethoxazole as a Viable Treatment Option for Infections Caused by Methicillin-ResistantStaphylococcus aureus Grim, Shellee A.; Rapp, Robert P.; Martin, Craig A. Pharmacotherapy, Vol. 25, Issue 2 https://doi.org/10.1592/phco.25.2.253.56956	journal	January 2005
The Antimicrobial Resistance Crisis: Causes, Consequences, and Management Michael, Carolyn Anne; Dominey-Howes, Dale; Labbate, Maurizio Frontiers in Public Health, Vol. 2 https://doi.org/10.3389/fpubh.2014.00145	journal	September 2014
Global disease burden due to antibiotic resistance – state of the evidence Woolhouse, Mark; Waugh, Catriona; Perry, Meghan Rose Journal of Global Health, Vol. 6, Issue 1 https://doi.org/10.7189/jogh.06.010306	journal	June 2016