Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds

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dc.contributor.authorDye, Keane J.en
dc.contributor.authorVogelaar, Nancy J.en
dc.contributor.authorO'Hara, Meganen
dc.contributor.authorSobrado, Pabloen
dc.contributor.authorSantos, Websteren
dc.contributor.authorCarlier, Paul R.en
dc.contributor.authorYang, Zhaominen
dc.date.accessioned2023-04-20T13:11:36Zen
dc.date.available2023-04-20T13:11:36Zen
dc.date.issued2022-12en
dc.description.abstractMany bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics.IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.en
dc.description.notesThis work was partially supported by the National Science Foundation grants MCB-1417726 and MCB-1919455 and a Lay Nam Chang Dean's Discovery Fund to Z.Y. K.J.D. was the recipient of a GSDA and the Lewis Edward Goyette Fellowship, as well as the Liberati Scholarship from Virginia Tech. We thank Kurt Piepenbrink for providing bacterial strains. We acknowledge Andreas Sukmana for his early contribution to the implementation of the HTS. K.J.D., N.J.V., P.S., and Z.Y. designed research and analyzed data. K.J.D. and N.J.V. performed experiments. M.O. aided in figure preparations. W.S. and P.R.C. provided analysis of HTS results. K.J.D. and Z.Y. wrote the manuscript. There are no conflicts of interest to declare.en
dc.description.sponsorshipNational Science Foundation [MCB-1417726, MCB-1919455]; Lay Nam Chang Dean's Discovery Fund; GSDA; Lewis Edward Goyette Fellowship; Liberati Scholarship from Virginia Techen
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1128/spectrum.03877-22en
dc.identifier.pmid36377931en
dc.identifier.urihttp://hdl.handle.net/10919/114588en
dc.language.isoenen
dc.publisherAmerican Society for Microbiologyen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectantivirulenceen
dc.subjectPilB ATPaseen
dc.subjecttype IV pilusen
dc.subjectT4P assemblyen
dc.subjecthigh-throughput screenen
dc.subjectbenserazideen
dc.subjectlevodopaen
dc.titleDiscovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compoundsen
dc.title.serialMicrobiology Spectrumen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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