A novel σB regulatory module in staphylococcus aureus: Unraveling the multifaceted roles of RsbU domains in stress response mechanisms

• Published in: Journal of microbiology, immunology and infection
• Main Authors: Huang, Yi-Hsi, Yeh, Wen-Bin, Chang, Renin, Chen, Chien-Yen, Wing-Yan Chan, Michael, Chou, Mei-Chia, Chen, Chien-Cheng
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 02.06.2025
• Subjects: Bacterial two-hybrid; Protein-protein interaction; RsbU; Staphylococcus aureus; RsbU; Protein-protein interaction; Bacterial two-hybrid; Staphylococcus aureus; σB; σ(B)
• ISSN: 1684-1182; 1995-9133; 1995-9133
• DOI: 10.1016/j.jmii.2025.05.007

The σB factor in Staphylococcus aureus governs the environmental stress response and a wide spectrum of biological functions. σB activity is regulated by protein-protein interactions among RsbU, RsbV, RsbW, and σB. While the C-terminal PP2C phosphatase domain of RsbU is well-characterized, the function of its N-terminal domain remains unclear. To analyze the molecular weight distributions of Rsb proteins and RsbV phosphorylation states, S. aureus cell lysates were subjected to gel filtration and Phos-tag gel electrophoresis. Protein associations were investigated through coelution experiments, immunoprecipitation, and a bacterial two-hybrid assay. Gel filtration revealed a shift in RsbV phosphorylation states following stress, with unphosphorylated monomeric RsbV predominating before stress and phosphorylated RsbV increasing afterward. This shift corresponded with a decrease in RsbV's ability to sequester RsbW. Under unstressed conditions, RsbU exhibited unexpectedly high phosphatase activity; however, unphosphorylated RsbV remained inactive in sequestering RsbW. Coelution and immunoprecipitation experiments demonstrated potential associations among RsbU, RsbW, and σB. The bacterial two-hybrid assay showed direct interactions between full-length RsbU and RsbV, while RsbU interacted with RsbW only in the presence of both RsbV and σB. Further experiments identified the N-terminal domain of RsbU as mediating interactions with RsbW. These findings reveal a novel σB regulatory module in S. aureus that integrates interactions among the N- and C-terminal domains of RsbU and other Rsb proteins. This module differs from σB regulatory mechanisms described in other bacteria, advancing our understanding of stress response regulation in S. aureus.