{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:58:13Z","timestamp":1785434293786,"version":"3.56.0"},"posted":{"date-parts":[[2025,5,10]]},"group-title":"Microbiology","reference-count":62,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,10]],"date-time":"2025-05-10T00:00:00Z","timestamp":1746835200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R01AI177619"],"award-info":[{"award-number":["R01AI177619"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P41GM135018"],"award-info":[{"award-number":["P41GM135018"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01GM038784"],"award-info":[{"award-number":["R01GM038784"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Small-molecule screens can advance therapeutic discovery while yielding new insights into pathogen biology. Through a luminescence-based screen, we identified clinically approved dihydropyridines that reduced the fitness of the intracellular pathogen\n                  <jats:italic>Brucella ovis<\/jats:italic>\n                  within mammalian phagocytes. Given the established role of dihydropyridines as inhibitors of mammalian L-type calcium channels and our observation that drug treatment perturbed calcium and manganese levels in host phagocytes, we initially hypothesized a host-directed mechanism of action. However, dose-response assays in axenic medium revealed that these drugs can directly inhibit\n                  <jats:italic>B. ovis<\/jats:italic>\n                  growth. To investigate the genetic basis of\n                  <jats:italic>B. ovis<\/jats:italic>\n                  susceptibility to dihydropyridine treatment, we selected for mutants capable of growing in the presence of cilnidipine. Cilnidipine-resistant isolates carried single-base deletions in the\n                  <jats:italic>bepE<\/jats:italic>\n                  pseudogene that restored an open reading frame encoding an RND-family transporter subunit.\n                  <jats:italic>B. ovis<\/jats:italic>\n                  is an ovine venereal pathogen that has experienced significant pseudogenization in its recent evolutionary history. Frameshift mutations that restored\n                  <jats:italic>bepE<\/jats:italic>\n                  function in\n                  <jats:italic>B. ovis<\/jats:italic>\n                  increased its resistance not only to dihydropyridines but also to a broad range of membrane-disrupting agents, including bile acid. Conversely, deleting\n                  <jats:italic>bepE<\/jats:italic>\n                  in\n                  <jats:italic>Brucella abortus<\/jats:italic>\n                  , a related zoonotic species that retains an intact version of the gene, increased its sensitivity to bile acid in vitro and to cilnidipine in the intracellular niche. We conclude that\n                  <jats:italic>bepE<\/jats:italic>\n                  is a key determinant of chemical stress resistance in\n                  <jats:italic>Brucella<\/jats:italic>\n                  spp., and that its pseudogenization in\n                  <jats:italic>B. ovis<\/jats:italic>\n                  contributes to the documented hypersensitivity of this host-restricted lineage to chemical stressors.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Brucella<\/jats:italic>\n                    species are intracellular bacterial pathogens that infect diverse wildlife and livestock and can also cause disease in humans. We developed a high-throughput assay to identify compounds that reduce the intracellular fitness of\n                    <jats:italic>Brucella ovis<\/jats:italic>\n                    , a globally distributed pathogen of sheep. This screen identified two dihydropyridine-class hypertension drugs as inhibitors of\n                    <jats:italic>B. ovis<\/jats:italic>\n                    fitness within mammalian cells. While dihydropyridines are established inhibitors of calcium channels in mammals, our results revealed that they also directly inhibited\n                    <jats:italic>B. ovis<\/jats:italic>\n                    growth. The\n                    <jats:italic>B. ovis<\/jats:italic>\n                    genome contains over 300 pseudogenes, and we discovered that resistance to dihydropyridines and other chemical stressors can emerge through frameshift mutations that resurrect the function of\n                    <jats:italic>bepE<\/jats:italic>\n                    , a conserved pseudogene encoding a membrane transporter subunit. In other\n                    <jats:italic>Brucella<\/jats:italic>\n                    species,\n                    <jats:italic>bepE<\/jats:italic>\n                    remains intact, and we show that deleting this gene in the zoonotic pathogen\n                    <jats:italic>Brucella abortus<\/jats:italic>\n                    increases susceptibility to both dihydropyridine treatment and membrane-disrupting stress. Our study provides evidence that pseudogenes like\n                    <jats:italic>bepE<\/jats:italic>\n                    can serve as a latent reservoir of adaptive functions in\n                    <jats:italic>B. ovis<\/jats:italic>\n                    , including drug and stress resistance.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.05.10.653276","type":"posted-content","created":{"date-parts":[[2025,5,11]],"date-time":"2025-05-11T03:05:12Z","timestamp":1746932712000},"source":"Crossref","is-referenced-by-count":0,"title":["Reversion of a RND transporter pseudogene uncovers latent stress resistance in\n                  <i>Brucella ovis<\/i>"],"prefix":"10.64898","author":[{"given":"Thomas","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Microbiology, Genetics & Immunology, Michigan State University, East Lansing, MI, USA"},{"name":"College of Osteopathic Medicine, Michigan State University, East Lansing, MI, 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