Deciphering the molecular basis of the global emergence of non-tuberculous Mycobacteria as important human respiratory pathogens

Moreover, the assay was validated using a panel of known anti-mycobacterial drugs, reproducing efficacy profiles similar to those reported in the literature. Based on our results, the fluorescence dilution system is an effective high throughput assay to test anti-mycobacterial drugs and host directed therapies.<br><br>To dissect host responses to infection and identify potential targets for host-directed therapy, 10x single cell RNA sequencing (scRNA-seq) of M. avium infected THP-1 macrophages was performed, revealing for the first time the heterogeneity in the host response; i.e, several large cell clusters showing different degrees of an inflammatory response, as well as a small cluster of infected cells showing a strong type I interferon response, suggesting a dynamic interplay of the immune signals at the single cell level.<br>Based on the differentially expressed genes identified in the scRNA-seq analysis, small molecule host targeted inhibitors were used in the FD assay, which led to the identification of importazole, a nuclear transport inhibitor, as a compound that inhibits intracellular replication.

Further mechanistic studies showed that importazole has direct antibacterial activity against M. avium, causing a reversible increase in bacterial cell length and inhibition of replication, suggesting that the compound could inhibit bacterial cell division.<br><br>Together, this work established and successfully employed a new image-based screening system to measure replication and persistence of M. avium in combination with scRNA-seq of infected macrophages, identifing the drug importazole as new inhibitor for M. avium replication, opening new avenues for the development of new therapies for M. avium infections.<br><br><i>Thesis embargoed until 31 July 2027</i>.