The aggregation of mycobacterial cells in a definite order, forming microscopic structures that resemble cords, is known as cord formation, or cording, and is considered a virulence factor in the M ycobacterium tuberculosis complex and the species M ycobacterium marinum . In the 1950s, cording was related to a trehalose dimycolate lipid that, consequently, was named the cord factor. However, modern techniques of microbial genetics have revealed that cording can be affected by mutations in genes not directly involved in trehalose dimycolate biosynthesis. Therefore, questions such as “How does mycobacterial cord formation occur?” and “Which molecular factors play a role in cord formation?” remain unanswered. At present, one of the problems in cording studies is the correct interpretation of cording morphology. Using optical microscopy, it is sometimes difficult to distinguish between cording and clumping, which is a general property of mycobacteria due to their hydrophobic surfaces. In this work, we provide a new way to visualize cords in great detail using scanning electron microscopy, and we show the first scanning electron microscopy images of the ultrastructure of mycobacterial cords, making this technique the ideal tool for cording studies. This technique has enabled us to affirm that nonpathogenic mycobacteria also form microscopic cords. Finally, we demonstrate that a strong correlation exists between microscopic cords, rough colonial morphology, and increased persistence of mycobacteria inside macrophages.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein–protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.