Druggable redox pathways against M. abscessus in cystic fibrosis patient-derived airway organoids

Abstract

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. Both our knowledge on and models to investigate the host and bacterial determinants that drive Mabs pathology in CF patients remain rudimentary. Here, we evaluated whether the lung organoid technology from CF patients is appropriate for modelling Mabs infection and whether antioxidant treatment is a candidate therapeutic approach in the context of CF disease. We derived airway organoids (AOs) from lung biopsy of a CF patient and characterized these AO by assessing CF transmembrane conductance regulator (CFTR) function, mucus and reactive oxygen species (ROS) production, lipid peroxidation, and cell death. We microinjected smooth (S-) or rough (R-)Mabs in the lumen of AOs to evaluate its fitness, responses of AOs to infection, and treatment efficacy by colony forming unit assay, qPCR and microscopy. We show that CF patient-derived AOs exhibited low residual CFTR function, enhanced mucus accumulation, and increased oxidative stress, lipid peroxidation, and cell death at basal state. While in AOs, S Mabs formed biofilm, R Mabs formed cord serpentines and displayed a higher virulence. S and R Mabs replicated more efficiently in CF AOs than in AOs derived from healthy lung. Pharmacological activation of antioxidant pathways resulted in better control of Mabs growth. In conclusion, we have established CF patient-derived AOs as a suitable human system to decipher mechanisms of CF-enhanced respiratory infection by Mabs and confirmed antioxidant approaches as a potential host-directed strategy to improve Mabs infection control.

Efficacy and Mode of Action of a Direct Inhibitor of Mycobacterium abscessus InhA

“…Consistently, we found that NITD-916 was as active as RFB in CF patient-derived AO recapitulating the CF airway dysfunctions, such as thick mucus, and with increased susceptibility to M. abscessus infection. 45 Thus, our data emphasize an unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.…”

“…In this biological model where mycobacteria are injected in AO, M. abscessus was found to actively replicate over 7 days in the lumen, and this was accompanied by reduced expression of mucin genes, usually participating in the clearance of pulmonary pathogens . Moreover, AO derived from CF patients were characterized by low CFTR activity and enhanced mucus accumulation, and the subsequent injection of M. abscessus in these 3D structures revealed the production of biofilms formed by the S variant and serpentine cords formed by the R variant . Importantly, both variants replicated more efficiently in CF AO than in AO derived from healthy lungs, thus highlighting the relevance of this model to study the pathogenesis of M. abscessus and to test the therapeutic potential of compounds in a system that recapitulates a CF environment .…”

“…45 Importantly, both variants replicated more efficiently in CF AO than in AO derived from healthy lungs, thus highlighting the relevance of this model to study the pathogenesis of M. abscessus and to test the therapeutic potential of compounds in a system that recapitulates a CF environment. 45 As illustrated in Figure 4A, CF-derived AO were injected with the M. abscessus S and R variants expressing Wasabi, and drug treatment was commenced 2 h later with NITD-916 at 1.56 μg/mL or 15.6 μg/ mL and with RFB at 12.5 μg/mL, included as a control drug. After 3 days of treatment, fluorescence microscopy showed high bacterial loads in AO left untreated or exposed to 1.56 μg/mL NITD-916, while exposing the AO to the 15.6 μg/mL dose significantly reduced the green fluorescence signal, similarly to the RFB treatment (Figure 4B).…”

“…Organoids were derived from a lung biopsy of a cystic fibrosis heterozygous patient (G542X/1811 + 1.6kbA → G), as previously reported. 45 Organoid Infections. On the infection day, organoids were infected with M. abscessus S and R variants adjusted to OD 600 = 0.2.…”

Cell envelope remodeling requires high concentrations of biotin during Mycobacterium abscessus model lung infection

“…Many studies have scrutinized how M. abscessus behaves in phagocytic cells such as macrophages [29][30][31][32][33][34][35] , and a variety of animal models have been developed for M. abscessus infection [36][37][38][39][40][41][42][43] ; however, these systems often represent systemic or invasive disease, and typically do not recapitulate the extracellular, luminal niche occupied by M. abscessus during human lung infection. Tissue culture systems represent an alternative approach to model lung infection, and can be successfully applied to M. abscessus [44][45][46][47] . Thus, to isolate the role of the lung environment in shaping M. abscessus gene essentiality, we have adapted an air-liquid interface tissue culture model for M. abscessus infection and used genome-wide saturating transposon mutagenesis and sequencing (TnSeq) to identify genes essential for survival in the lung environment.…”