Reversion of a RND transporter pseudogene uncovers latent stress resistance in Brucella ovis

ABSTRACT 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 Brucella ovis 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 B. ovis growth. To investigate the genetic basis of B. ovis 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 bepE pseudogene that restored an open reading frame encoding an RND-family transporter subunit. B. ovis is an ovine venereal pathogen that has experienced significant pseudogenization in its recent evolutionary history. Frameshift mutations that restored bepE function in B. ovis increased its resistance not only to dihydropyridines but also to a broad range of membrane-disrupting agents, including bile acid. Conversely, deleting bepE in Brucella abortus , 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 bepE is a key determinant of chemical stress resistance in Brucella spp., and that its pseudogenization in B. ovis contributes to the documented hypersensitivity of this host-restricted lineage to chemical stressors. Author Summary Brucella 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 Brucella ovis , a globally distributed pathogen of sheep. This screen identified two dihydropyridine-class hypertension drugs as inhibitors of B. ovis fitness within mammalian cells. While dihydropyridines are established inhibitors of calcium channels in mammals, our results revealed that they also directly inhibited B. ovis growth. The B. ovis 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 bepE , a conserved pseudogene encoding a membrane transporter subunit. In other Brucella species, bepE remains intact, and we show that deleting this gene in the zoonotic pathogen Brucella abortus increases susceptibility to both dihydropyridine treatment and membrane-disrupting stress. Our study provides evidence that pseudogenes like bepE can serve as a latent reservoir of adaptive functions in B. ovis , including drug and stress resistance.