Engineering chaperone/usher pathway pili for surface display: Structural constraints, design principles, and...

Area of Science:

• Microbiology
• Structural Biology
• Synthetic Biology

Background:

• Chaperone/Usher (CU) pathway pili are diverse, highly abundant protein filaments on Gram-negative bacteria.
• Their repetitive structure and micrometer length offer potential for surface display applications.
• Previous engineering efforts were limited by a lack of structural and mechanistic understanding.

Purpose of the Study:

• To analyze structural constraints governing CU pilus display.
• To reinterpret past display strategies using a unified structural framework.
• To propose engineering approaches for versatile CU pilus-based display platforms.

Main Methods:

• Systematic analysis of structurally characterized CU pili from E. coli, Salmonella, and Y. pestis.
• Reinterpretation of existing display strategies through a structural lens.
• Distillation of conserved design principles for engineering permissiveness.

Main Results:

• Identification of key structural constraints influencing CU pilus display.
• Development of a unified structural framework for understanding pilus assembly and display.
• Distillation of conserved design principles for engineering CU pili.

Conclusions:

• CU pili engineering is significantly advanced by recent structural data.
• A structure-guided framework facilitates assessment and design of CU pilus display systems.
• This work provides a roadmap for expanding CU pili into tunable synthetic biology platforms.