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Respiratory infections remain a major global health concern. Tuberculosis is one of the top 10 causes of death worldwide, while infections with Non-Tuberculous Mycobacteria are rising globally. Recent advances in human tissue modeling offer a unique opportunity to grow different human "organs" in vitro, including the human airway, that faithfully recapitulates lung architecture and function. Here, we have explored the potential of human airway organoids (AOs) as a novel system in which to assess the very early steps of mycobacterial infection. We reveal that Mycobacterium tuberculosis (Mtb) and Mycobacterium abscessus (Mabs) mainly reside as extracellular bacteria and infect epithelial cells with very low efficiency. While the AO microenvironment was able to control, but not eliminate Mtb, Mabs thrives. We demonstrate that AOs responded to infection by modulating cytokine, antimicrobial peptide, and mucin gene expression. Given the importance of myeloid cells in mycobacterial infection, we co-cultured infected AOs with human monocyte-derived macrophages and found that these cells interact with the organoid epithelium. We conclude that adult stem cell (ASC)-derived AOs can be used to decipher very early events of mycobacteria infection in human settings thus offering new avenues for fundamental and therapeutic research., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro -starved Mycobacterium marinum In vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo To prolong the persister phenotype in vivo , we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the Δ rpfAB mutant, lacking two Rpfs, established an infection in vivo , whereas a nutrient-starved Δ rpfAB mutant did maintain its persister phenotype in vivo This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo , as would be expected for persisters. We propose that this zebrafish embryo model with Δ rpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters., (Copyright © 2020 American Society for Microbiology.)