In situ architecture of the Tad pilus machine in Caulobacter crescentus

The tight adherence (Tad) pilus is a broadly distributed and evolutionarily distinct subclass of type IV pili that mediate cell adhesion, biofilm formation, predation, and surface sensing in many bacteria, including Caulobacter crescentus , Myxococcus xanthus , Vibrio vulnificus , and Bifidobacterium breve . Tad pili undergo cycles of extension and retraction powered by a cell-envelope-embedded nanomachine. Despite their biological importance, the architecture and assembly mechanism of the Tad pilus system remain poorly understood. Although cryo-electron tomography (cryo-ET) has elucidated the in situ architectures of other type IV pilus systems, no intact Tad machine structure has previously been reported. Here, we use cryo-ET and subtomogram averaging to resolve the architecture of the C. crescentus Tad pilus within the bacterial cell envelope. A three-dimensional classification further reveals multiple assembly intermediates, and integrative modeling incorporating AlphaFold3 predictions helps define the spatial arrangement of all core components. The resulting structural framework gives insight into the stepwise assembly process of the C. crescentus Tad pilus machine. Altogether, our results provide an in situ architectural model of the Tad pilus machine, establishing a foundation for understanding homologous systems across a broad range of bacteria. IMPORTANCE Investigating the Tad pilus nanomachine in a genetically tractable, non-pathogenic organism like Caulobacter crescentus provides a powerful model for elucidating the architecture and functional dynamics of this widespread system. Insights gained from studying the Tad machinery can improve our understanding of related Tad pilus systems in pathogenic bacteria such as Aggregatibacter actinomycetemcomitans , where Tad pili are a key determinant of biofilm formation and chronic infection. Additionally, the remarkable functional diversity of Tad systems, ranging from surface sensing in C. crescentus to bacterial predation in Myxococcus xanthus , highlights their broad biological relevance. By revealing the in situ architecture of the Tad pilus biosynthetic machinery, this study advances our understanding of a major class of bacterial nanomachines and may thus provide structural insights that could inform the development of new therapeutic strategies targeting pilus-mediated virulence.