Kim Thomas, Hong Bongjin, O'Halloran Thomas V, Lisabeth Erika, Neubig Richard R, Fiebig Aretha, Crosson Sean
Department of Microbiology, Genetics & Immunology, Michigan State University, East Lansing, MI, USA.
College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA.
bioRxiv. 2025 May 10:2025.05.10.653276. doi: 10.1101/2025.05.10.653276.
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 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 growth. To investigate the genetic basis of 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 pseudogene that restored an open reading frame encoding an RND-family transporter subunit. is an ovine venereal pathogen that has experienced significant pseudogenization in its recent evolutionary history. Frameshift mutations that restored function in increased its resistance not only to dihydropyridines but also to a broad range of membrane-disrupting agents, including bile acid. Conversely, deleting in , 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 is a key determinant of chemical stress resistance in spp., and that its pseudogenization in contributes to the documented hypersensitivity of this host-restricted lineage to chemical stressors.
小分子筛选可以推动治疗方法的发现，同时为病原体生物学提供新的见解。通过基于发光的筛选，我们鉴定出临床批准的二氢吡啶类化合物，它们能降低哺乳动物吞噬细胞内细胞内病原体的适应性。鉴于二氢吡啶类化合物作为哺乳动物L型钙通道抑制剂的既定作用，以及我们观察到药物治疗会扰乱宿主吞噬细胞中的钙和锰水平，我们最初假设其作用机制是针对宿主的。然而，在无菌培养基中的剂量反应试验表明，这些药物可以直接抑制其生长。为了研究对二氢吡啶治疗敏感性的遗传基础，我们筛选出了能够在西尼地平存在下生长的突变体。对西尼地平耐药的分离株在假基因中携带单碱基缺失，该缺失恢复了一个编码RND家族转运蛋白亚基的开放阅读框。是一种绵羊性病原体，在其最近的进化历史中经历了显著的假基因化。在中恢复功能的移码突变不仅增加了其对二氢吡啶类化合物的抗性，还增加了对包括胆汁酸在内的多种膜破坏剂的抗性。相反，在保留该基因完整版本的相关人畜共患病物种中删除该基因，增加了其在体外对胆汁酸和在细胞内环境中对西尼地平的敏感性。我们得出结论，是 spp. 中化学应激抗性的关键决定因素，并且它在中的假基因化导致了这个宿主受限谱系对化学应激源的超敏反应。
