Three-dimensional in vivo monitoring of mycobacterial infections and therapeutic efficacy based on tissue-clearing technology CUBIC

Abstract

Abstract Mycobacteria are a continuous threat to human health. They include various species, such as Mycobacterium tuberculosis (M. tuberculosis), which is an intracellular parasite of mammals, and the most virulent and non-tuberculous mycobacteria (NTM), namely, M. avium, which are environmental bacteria causing intractable NTM diseases. An infection model of transparent zebrafish and fish-infectious M. marinum was established to better understand the in vivo behavior of mycobacteria under the pressure of host immune responses. However, the fish model does not fully replicate mammalian immunity. Here, we demonstrate that a clear, unobstructed brain/body imaging cocktail and computational analysis (CUBIC)-based infection (CUBIC-infection) analysis enables comprehensive mycobacterial profiling of the whole lung. We assessed the in vivo kinetics of mycobacterial infection along with fluorescent protein-expressing recombinant mycobacteria. We detected mycobacterium at a single bacterial level and counted bacterial numbers, which was comparable to the colony-forming units of organ homogenates. CUBIC-infection analysis distinguished in vivo spatiotemporal behavior of M. tuberculosis, M. tuberculosisvariant Bacillus Calmette-Guerin, and M. avium in mice. Furthermore, it monitored spatiotemporal information on the therapeutic efficacies of anti-tuberculosis drugs and an anti-lymphangiogenesis agent. Our data suggest that CUBIC-infection analysis is a powerful tool for understanding mycobacterial infections in mammals and developing therapeutic agents.