# Reversion of a RND transporter pseudogene reveals latent stress resistance potential in Brucella ovis

## TL;DR

A study shows that restoring a pseudogene in Brucella ovis increases its resistance to drugs and stressors, revealing hidden adaptive potential in this pathogen.

## Contribution

The study reveals that reactivating a pseudogenized RND transporter gene in Brucella ovis confers broad stress resistance through mutational reversion.

## Key findings

- BepE pseudogene reversion in Brucella ovis increases resistance to dihydropyridines and cell envelope disruptors.
- BepE deletion in Brucella abortus increases sensitivity to dihydropyridines and membrane-disrupting agents.
- Pseudogenes like bepE serve as latent reservoirs of adaptive potential in Brucella species.

## Abstract

Small-molecule screens can advance therapeutic discovery and uncover new features of pathogen biology. Through a luminescence-based screen, we identified clinically approved dihydropyridines that impaired fitness of the intracellular pathogen Brucella ovis in mammalian phagocytes. Given that dihydropyridines block mammalian L-type calcium channels, and based on 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 showed that dihydropyridines have direct antimicrobial effects. To explore the genetic basis of dihydropyridine sensitivity, we selected for B. ovis mutants capable of growing in the presence of cilnidipine, a representative compound from this drug class. Cilnidipine-resistant mutants harbored single-nucleotide deletions in the bepE transporter pseudogene that restored its open reading frame, enabling expression of a functional RND-family transporter. B. ovis is a host-restricted ovine pathogen that has experienced significant pseudogenization in its recent evolutionary history. Reversion mutations that restored the open reading frame of the bepE pseudogene increased B. ovis resistance not only to dihydropyridines but also to a broad range of cell envelope-disrupting agents. Conversely, deleting bepE in Brucella abortus, a closely related zoonotic species that retains an intact version of the gene, increased its sensitivity to envelope disruptors 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.

Brucella species are intracellular bacterial pathogens that infect diverse wildlife and livestock and can also cause disease in humans. We developed a high-throughput screen to discover compounds that reduced the intracellular fitness of Brucella ovis. The screen identified dozens of molecules that impaired Brucella survival within its host cell niche, including two dihydropyridine-class antihypertensive drugs. Dihydropyridines are well-established inhibitors of mammalian calcium channels, and our study provides evidence that they reduce intracellular B. ovis fitness through both host-targeted and direct antibacterial mechanisms. The B. ovis genome is extensively pseudogenized, and we discovered that resistance to dihydropyridines and other chemical stressors can arise through frameshift mutations that rescue the function of bepE, a conserved RND-family transporter pseudogene. bepE remains intact and functional in other Brucella species, and deletion of this gene in the zoonotic pathogen Brucella abortus increases its sensitivity to both dihydropyridine treatment and membrane-disrupting compounds. Our study shows that pseudogenes like bepE serve as a latent reservoir of adaptive potential in B. ovis, enabling stress resistance through mutational reactivation.