Lormand Justin D, Savelle Charles H, Teschler Jennifer K, López Eva, Little Richard H, Malone Jacob G, Yildiz Fitnat H, García-García María J, Sondermann Holger
CSSB Centre for Structural Systems Biology, Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany.
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
mBio. 2025 Jun 3:e0087225. doi: 10.1128/mbio.00872-25.
Proteases regulate important biological functions. Here, we present the structural and functional characterization of three previously uncharacterized aspartic proteases in . We show that these proteases have structural hallmarks of retropepsin peptidases and play redundant roles for cell survival under hypoosmotic stress conditions. Consequently, we named them retropepsin-like osmotic stress tolerance peptidases (Rlo). Our research shows that while Rlo proteases are homologous to RimB, an aspartic peptidase involved in rhizosphere colonization and plant infection, they contain N-terminal signal peptides and perform distinct biological functions. Mutants lacking all three secreted Rlo peptidases show defects in antibiotic resistance, biofilm formation, and cell morphology. These defects are rescued by mutations in the inactive transglutaminase transmembrane protein RloB and the cytoplasmic ATP-grasp protein RloC, two previously uncharacterized genes in the same operon as one of the Rlo proteases. These studies identify Rlo proteases and operon products as critical factors in clinically relevant processes, making them appealing targets for therapeutic strategies against infections.IMPORTANCEBacterial infections have become harder to treat due to the ability of pathogens to adapt to different environments and the rise of antimicrobial resistance. This has led to longer illnesses, increased medical costs, and higher mortality rates. The opportunistic pathogen is particularly problematic because of its inherent resistance to many antibiotics and its capacity to form biofilms, structures that allow bacteria to withstand hostile conditions. Our study uncovers a new class of retropepsin-like proteases in that are required for biofilm formation and bacterial survival under stress conditions, including antibiotic exposure. By identifying critical factors that determine bacterial fitness and adaptability, our research lays the foundation for developing new therapeutic strategies against bacterial infections.
蛋白酶调节重要的生物学功能。在此，我们展示了三种先前未被表征的天冬氨酸蛋白酶的结构和功能特征。我们表明，这些蛋白酶具有逆转胃蛋白酶肽酶的结构特征，并且在低渗应激条件下对细胞存活发挥冗余作用。因此，我们将它们命名为逆转胃蛋白酶样渗透应激耐受肽酶（Rlo）。我们的研究表明，虽然Rlo蛋白酶与参与根际定殖和植物感染的天冬氨酸肽酶RimB同源，但它们含有N端信号肽并执行不同的生物学功能。缺乏所有三种分泌型Rlo肽酶的突变体在抗生素抗性、生物膜形成和细胞形态方面表现出缺陷。这些缺陷通过无活性转谷氨酰胺酶跨膜蛋白RloB和细胞质ATP结合蛋白RloC中的突变得以挽救，这两个基因是与其中一种Rlo蛋白酶位于同一操纵子中的两个先前未被表征的基因。这些研究确定Rlo蛋白酶和操纵子产物是临床相关过程中的关键因素，使其成为针对感染的治疗策略的有吸引力的靶点。重要性由于病原体适应不同环境的能力以及抗菌药物耐药性的上升，细菌感染变得更难治疗。这导致病程延长、医疗成本增加和死亡率上升。机会性病原体尤其成问题，因为它对许多抗生素具有固有抗性，并且能够形成生物膜，这种结构使细菌能够抵御恶劣条件。我们的研究揭示了中的一类新的逆转胃蛋白酶样蛋白酶，它们是生物膜形成和细菌在包括抗生素暴露在内的应激条件下存活所必需的。通过确定决定细菌适应性和生存能力的关键因素，我们的研究为开发针对细菌感染的新治疗策略奠定了基础。
