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

Chaperone/Usher (CU) pathway pili constitute the most diverse class of pili in Gram-negative bacteria. Each pilus filament is assembled from thousands of pilin subunits, and thousands of such filaments can be simultaneously displayed on the bacterial surface, where they mediate essential functions related to environmental adaptation. Owing to their distinctive architectural features, including micrometer-scale length, high copy number, and repetitive subunit organization, CU pili possess intrinsic potential as platforms for bacterial surface display in biotechnology and synthetic biology. Early efforts in the 1980s and 1990s explored CU pili as display scaffolds but failed to yield broadly applicable or robust systems, largely due to limited understanding of pilus assembly mechanisms and the absence of high-resolution structural information. In recent years, the rapid accumulation of atomic and near-atomic resolution structures of CU pilins and their assembly intermediates, together with advances in protein structure prediction, has fundamentally reshaped the landscape of pilus engineering. In this review, we use structurally characterized CU pili from Escherichia coli, Salmonella and Yersinia pestis as reference models to systematically analyze the structural constraints governing CU pilus-based display. We reinterpret prior display strategies through a unified structural framework, distill conserved design principles that define engineering permissiveness, and propose practical engineering approaches for expanding CU pili into versatile and tunable surface display platforms. Collectively, this work provides a structure-guided framework for assessing feasibility and guiding the rational engineering of CU pilus display systems within the synthetic biology toolbox.