EccC3 is essential for pathogenesis of rough Mycobacterium abscessus in zebrafish

Mycobacterium abscessus (Mab) is an emerging human pathogen causing severe lung infections in cystic fibrosis patients and displays either a smooth (S) or a rough (R) morphotype, the latter being associated with a strong inflammatory response. In contrast to most other mycobacteria, it possesses only two ESX secretion systems, ESX-3 and ESX-4. Recent work showed that infection of Mab S lacking the eccC3-encoding ATPase component of ESX-3 in mice was associated with reduced bacterial loads in lungs and no mortality. While these studies emphasize the importance of ESX-3 in the virulence of Mab S, its contribution to the pathogenesis of Mab R in vivo remains undefined. Here, we exploited an Mab R mutant lacking eccC3 using the zebrafish model, particularly suited to describe the chronology and pathophysiological signs of Mab R infection. Our results clearly show that R ΔeccC3 was highly attenuated in larvae, as evidenced by the enhanced larvae survival, the reduced bacterial burden, cord number, and limited proportion of abscesses, while functional complementation of eccC3 partially restored the phenotypes to wild-type levels. The phenotypes were corroborated with whole larvae imaging, showing lower bacterial foci in R ΔeccC3, as compared to the parental R strain. Moreover, infection with R ΔeccC3 was associated with a lower pro-inflammatory response, as showcased by the reduced production of il1b and tnfa transcripts. Together, these findings demonstrate that ESX-3 is a primary driver of Mab R pathogenicity. IMPORTANCE Mycobacterium abscessus (Mab) is currently recognized as an extremely difficult-to-manage pathogen in patients with cystic fibrosis. The rough (R) morphotype of Mab is associated with chronic and more aggressive infections in patients than the smooth (S) morphotype. However, the mechanisms of pathogenesis of Mab R remain largely unexplored. In this study, we investigated the infection capacity and virulence of a Mab R mutant lacking a functional ESX-3 secretion system using the zebrafish model. The mutant was severely impaired in its ability to replicate in the larvae, and this was correlated with a significant increase in larvae survival, highlighting the critical role of ESX-3 in Mab R virulence. Overall, this emphasizes ESX-3 as an attractive drug target for future drug developments against Mab R lung diseases.