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An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy

Bulterys, Philip L. and Toesca, Isabelle J. and Norris, Michael H. and Maloy, Jeffrey P. and Fitz-Gibbon, Sorel T. and France, Bryan and Toffig, Babak and Morselli, Marco and Somprasong, Nawarat and Pellegrini, Matteo and Schweizer, Herbert P. and Tuanyok, Apichai and Damoiseaux, Robert and French, Christopher T. and Miller, Jeff F. (2019) An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy. Proceedings of the National Academy of Sciences of the United States of America, 116 (37). pp. 18597-18606. ISSN 0027-8424. PMCID PMC6744847. https://resolver.caltech.edu/CaltechAUTHORS:20190903-100027406

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Abstract

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm. Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell–cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia. In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.1906388116DOIArticle
https://www.pnas.org/content/suppl/2019/08/21/1906388116.DCSupplementalPublisherSupporting Information
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc6744847/PubMed CentralArticle
ORCID:
AuthorORCID
Fitz-Gibbon, Sorel T.0000-0001-7090-5719
Additional Information:© 2019 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Contributed by Jeff F. Miller, July 13, 2019 (sent for review April 15, 2019; reviewed by Yunn-Hwen Gan and John J. Mekalanos). We thank the members of the J.F.M. laboratory for valuable discussions. We thank undergraduate researchers Minna Ding, Christy Kim, and Anh Huynh (UCLA) for their technical assistance. P.L.B. thanks Steve Smale, Kelsey Martin, Robert Modlin, Marcus Horwitz, Alex Hoffmann, Kent Hill, Ann Hirsch, Elizabeth Neufeld, Atish Ganguly, and Umesh Ahuja for their mentorship. We thank Bart Currie and his group at the Menzies School of Health Research for providing the MSHR305 Bp strain. We thank Romney Humphries and the UCLA Clinical Microbiology Laboratory for providing P. aeruginosa and E. coli strains. We also thank Colin Manoil (University of Washington) for providing us with the Bt transposon mutant library. We thank Ken Ng for his artistic guidance and Alexander Sun (California Institute of Technology [Caltech]) for his organic chemistry expertise. This work was supported by the NIH Pacific Southwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases under award U54 AI065359 to J.F.M. and the Defense Threat Reduction Agency under awards HDTRA1-11-1-003 and HDTRA1-17-1-0015 to J.F.M. and C.T.F. P.L.B. received fellowship support from the National Institute of Allergy and Infectious Diseases of the NIH (F30AI118342), the UCLA–Caltech Medical Scientist Training Program (T32GM008042), and a Paul and Daisy Soros Fellowship for New Americans. H.P.S. was supported by University of Florida Preeminence start-up funds. Animal studies were funded by Emerging Pathogens Institute seed fund award 16-3 to M.H.N. Author contributions: P.L.B., I.J.T., R.D., C.T.F., and J.F.M. designed research; P.L.B., I.J.T., M.H.N., S.T.F.-G., B.F., B.T., and M.M. performed research; I.J.T., M.H.N., J.P.M., S.T.F.-G., B.F., B.T., M.M., N.S., M.P., H.P.S., A.T., R.D., C.T.F., and J.F.M. contributed new reagents/analytic tools; P.L.B., I.J.T., M.H.N., S.T.F.-G., C.T.F., and J.F.M. analyzed data; and P.L.B., C.T.F., and J.F.M. wrote the paper. Reviewers: Y.-H.G., National University of Singapore; and J.J.M., Harvard University. The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1906388116/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
NIHU54 AI065359
Defense Threat Reduction Agency (DTRA)HDTRA1-11-1-003
Defense Threat Reduction Agency (DTRA)HDTRA1-17-1-0015
NIH Postdoctoral FellowshipF30AI118342
UCLA–Caltech Medical Scientist Training ProgramUNSPECIFIED
NIH Predoctoral FellowshipT32GM008042
Paul and Daisy Soros FellowshipUNSPECIFIED
University of FloridaUNSPECIFIED
Emerging Pathogens Institute16-3
Subject Keywords:Burkholderia pseudomallei; melioidosis; type 6 secretion system (T6SS); small molecule; drug discovery
Issue or Number:37
PubMed Central ID:PMC6744847
Record Number:CaltechAUTHORS:20190903-100027406
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190903-100027406
Official Citation:An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy. Philip L. Bulterys, Isabelle J. Toesca, Michael H. Norris, Jeffrey P. Maloy, Sorel T. Fitz-Gibbon, Bryan France, Babak Toffig, Marco Morselli, Nawarat Somprasong, Matteo Pellegrini, Herbert P. Schweizer, Apichai Tuanyok, Robert Damoiseaux, Christopher T. French, Jeff F. Miller. Proceedings of the National Academy of Sciences Sep 2019, 116 (37) 18597-18606; DOI: 10.1073/pnas.1906388116
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:98382
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:03 Sep 2019 18:20
Last Modified:02 Jun 2020 18:35

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