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Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms

VanDrisse, Chelsey M. and Lipsh-Sokolik, Rosalie and Khersonsky, Olga and Fleishman, Sarel J. and Newman, Dianne K. (2021) Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms. Proceedings of the National Academy of Sciences of the United States of America, 118 (12). Art. No. e2022012118. ISSN 0027-8424. PMCID PMC8000102. https://resolver.caltech.edu/CaltechAUTHORS:20210316-072135929

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Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA’s obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.2022012118DOIArticle
https://www.pnas.org/content/suppl/2021/03/13/2022012118.DCSupplementalPublisherSupporting Information
ORCID:
AuthorORCID
VanDrisse, Chelsey M.0000-0001-9139-0443
Lipsh-Sokolik, Rosalie0000-0002-5548-7309
Fleishman, Sarel J.0000-0003-3177-7560
Newman, Dianne K.0000-0003-1647-1918
Additional Information:© 2021 National Academy of Sciences. Published under the PNAS license. Edited by David Baker, University of Washington, Seattle, WA, and approved February 16, 2021 (received for review October 29, 2020). This work was supported by the Schwartz/Reisman Collaborative Science Program; NIH Grants 1R01AI127850-01A1 and 1R01HL152190-01; and the Doren Family Foundation. Research in the S.J.F. laboratory was further supported by the Dr. Barry Sherman Institute for Medicinal Chemistry; a Consolidator Grant from the European Research Council (815379); and the Israel Science Foundation (1844). R.L.-S. was supported by a fellowship from the Arianne de Rothschild Women Doctoral Program. We thank Steven Wilbert and Melanie Spero for assistance with ABBA experiments; Steven Wilbert for image analysis; John Ciemniecki for image and statistical analyses; Louise Siskel for experimental assistance; and Scott Saunders for providing growth curve fitting software. Data Availability: All study data are included in the article and/or supporting information. Author contributions: C.M.V., R.L.-S., O.K., S.J.F., and D.K.N. designed research; C.M.V., R.L.-S., and O.K. performed research; C.M.V., R.L.-S., O.K., S.J.F., and D.K.N. analyzed data; and C.M.V., R.L.-S., O.K., S.J.F., and D.K.N. wrote the paper. Competing interest statement: C.M.V., R.L.-S., O.K., S.J.F., and D.K.N. are named inventors on patents filed by Caltech and the Weizmann Institute on the design methods. This article is a PNAS Direct Submission. This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2022012118/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
Schwartz/Reisman Collaborative Science ProgramUNSPECIFIED
NIH1R01AI127850-01A1
NIH1R01HL152190-01
Doren Family FoundationUNSPECIFIED
Dr. Barry Sherman Institute for Medicinal ChemistryUNSPECIFIED
European Research Council (ERC)815379
Israel Science Foundation1844
Arianne de Rothschild Women Doctoral ProgramUNSPECIFIED
Subject Keywords:Pseudomonas aeruginosa; biofilms; antibiotic tolerance; pyocyanin; AffiLib
Issue or Number:12
PubMed Central ID:PMC8000102
Record Number:CaltechAUTHORS:20210316-072135929
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210316-072135929
Official Citation:Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms. Chelsey M. VanDrisse, Rosalie Lipsh-Sokolik, Olga Khersonsky, Sarel J. Fleishman, Dianne K. Newman. Proceedings of the National Academy of Sciences Mar 2021, 118 (12) e2022012118; DOI: 10.1073/pnas.2022012118
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108445
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:19 Mar 2021 02:06
Last Modified:19 Apr 2021 21:44

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