In situ NMR reveals a pH sensor motif in an outer membrane protein that drives bacterial vesicle production

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Outer membrane vesicles (OMVs) are crucial for bacterial functions like homeostasis, secretion, communication, and pathogenesis.
• Salmonella enterica Typhimurium (STm) upregulates the outer membrane protein PagC within acidic host cell vacuoles to activate OMV biogenesis.
• PagC is a highly expressed gene in host environments, indicating its significant role in STm pathogenesis.

Purpose of the Study:

• To investigate the pH-sensing mechanism of the outer membrane protein PagC involved in OMV biogenesis.
• To elucidate the structural basis of PagC's function in OMV production and aggregation.
• To explore the role of PagC in bacterial adhesion and biofilm formation.

Main Methods:

• Solid-state nuclear magnetic resonance (NMR) spectroscopy was employed to study PagC within native bacterial OMVs.
• Electron microscopy (EM) was used to visualize OMV aggregation and bacterial cell morphology.
• Functional assays were performed in Escherichia coli to replicate and study PagC's role in OMV production and pellicle formation.

Main Results:

• Three histidine residues in PagC were identified as essential for its OMV biogenic function and act as a pH-sensing motif.
• NMR data revealed that PagC undergoes protonation around pH 6, with associated structural changes in specific regions.
• PagC expression enhanced OMV production and induced OMV aggregation at acidic pH, also increasing bacterial pellicle formation.

Conclusions:

• PagC functions as a pH sensor, regulating OMV biogenesis and aggregation in response to acidic environments.
• The study provides structural insights into PagC's mechanism of action using native OMVs, offering a physiologically relevant context.
• PagC may play a role as an adhesin involved in bacterial biofilm formation under acidic conditions.