High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB

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dc.contributor.authorDye, Keane J.en
dc.contributor.authorVogelaar, Nancy J.en
dc.contributor.authorSobrado, Pabloen
dc.contributor.authorYang, Zhaominen
dc.contributor.departmentBiological Sciencesen
dc.contributor.departmentCenter for Drug Discoveryen
dc.contributor.departmentBiochemistryen
dc.date.accessioned2021-05-05T15:33:44Zen
dc.date.available2021-05-05T15:33:44Zen
dc.date.issued2021-03en
dc.description.abstractThe bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro. Chloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems. IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.en
dc.description.notesThis work was partially supported by the Fralin Life Science Institute and National Science Foundation grants MCB-1417726 and MCB-1919455 to Z.Y. K.J.D. is the recipient of a GSDA fellowship and the Liberati Scholarship. We would like to acknowledge the Open Access Subvention Fund (OASF) of Virginia Tech for its support in the publication of this article. K.J.D., N.J.V., and Z.Y. designed research and analyzed data. K.J.D. and N.J.V. performed experiments. P.S. provided guidelines for HTS. K.J.D. and Z.Y. wrote the manuscript.?; We thank Andreas Sukamana for conducting some of the early experiments for the work reported here. There is no conflict of interest to declare.en
dc.description.sponsorshipFralin Life Science Institute; National Science FoundationNational Science Foundation (NSF) [MCB-1417726, MCB-1919455]; GSDA fellowship; Liberati Scholarship; Open Access Subvention Fund (OASF) of Virginia Techen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1128/mSphere.00129-21en
dc.identifier.issn2379-5042en
dc.identifier.issue2en
dc.identifier.othere00129-21en
dc.identifier.pmid33658276en
dc.identifier.urihttp://hdl.handle.net/10919/103192en
dc.identifier.volume6en
dc.language.isoenen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectPilB ATPaseen
dc.subjecttype IV pili (T4P)en
dc.subjecthigh-throughput screen (HTS)en
dc.subjectantivirulenceen
dc.subjectquercetinen
dc.subjectmotilityen
dc.subjectMyxococcus xanthusen
dc.titleHigh-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilBen
dc.title.serialmSphereen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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