A functional amyloid matrix underpins the PDIM-architected corded superstructure of the Mycobacterium tuberculosis biofilm.

Mycobacterium tuberculosis (Mtb) biofilm formation is associated with antibiotic tolerance, but its architecture remains poorly understood. Here, we reveal that these biofilms form highly-organized superstructures of cords, and through their deconstruction, provide a new molecular insight into Mtb biofilms. Using multimodal imaging, we demonstrate that the lipid Phthiocerol Dimycocerosate (PDIM) is required for organizing bacilli into foundational cords and contributes specifically to biofilm-associated antibiotic tolerance. In contrast, the ESX-1 secretion system enhances the biochemical complexity of the extracellular matrix. Notably, we identified a functional amyloid matrix that encases bacterial cords or aggregates within the biofilm, likely conferring structural integrity. Together, these findings support a three-component model that distinguishes structural integrity, physical organization, and biochemical maturation, establishing a new architectural framework for Mtb biofilms. Finally, we show that the natural compound epigallocatechin gallate (EGCG) disrupts biofilm formation, highlighting the therapeutic potential of targeting this architecture to overcome drug tolerance in tuberculosis.