EccC3 is essential for pathogenesis of rough <i>Mycobacterium abscessus</i> in zebrafish

<i>Mycobacterium abscessus</i> (<i>Mab</i>) 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 <i>Mab</i> S lacking the <i>eccC3</i>-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 <i>Mab</i> S, its contribution to the pathogenesis of <i>Mab</i> R <i>in vivo</i> remains undefined. Here, we exploited an <i>Mab</i> R mutant lacking <i>eccC3</i> using the zebrafish model, particularly suited to describe the chronology and pathophysiological signs of <i>Mab</i> R infection. Our results clearly show that R Δ<i>eccC3</i> 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 <i>eccC3</i> partially restored the phenotypes to wild-type levels. The phenotypes were corroborated with whole larvae imaging, showing lower bacterial foci in R Δ<i>eccC3</i>, as compared to the parental R strain. Moreover, infection with R Δ<i>eccC3</i> was associated with a lower pro-inflammatory response, as showcased by the reduced production of <i>il1b</i> and <i>tnfa</i> transcripts. Together, these findings demonstrate that ESX-3 is a primary driver of <i>Mab</i> R pathogenicity. IMPORTANCE <i>Mycobacterium abscessus</i> (<i>Mab</i>) is currently recognized as an extremely difficult-to-manage pathogen in patients with cystic fibrosis. The rough (R) morphotype of <i>Mab</i> is associated with chronic and more aggressive infections in patients than the smooth (S) morphotype. However, the mechanisms of pathogenesis of <i>Mab</i> R remain largely unexplored. In this study, we investigated the infection capacity and virulence of a <i>Mab</i> 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 <i>Mab</i> R virulence. Overall, this emphasizes ESX-3 as an attractive drug target for future drug developments against <i>Mab</i> R lung diseases.