Comparative genomics and molecular insights into smooth and rough clinical isolates of Mycobacterium abscessus

Background. Our recent findings indicate that the rough form of the Mycobacterium abscessus complex (MABC) is associated with worse clinical outcomes compared to the smooth variant. The glycopeptidolipid (GPL) locus has been considered vital for this transition; however, this has only been shown during in vitro selection. Thus, we aimed to investigate genetic differences between the morphotypes among clinical isolates. Methods. Whole-genome sequencing (WGS) by Oxford Nanopore was applied to rough and smooth clinical MABC strains ( n =10), including laboratory strains and in vitro exposure to nitric oxide (NO). Comparative genomic analysis, methylation and pangenome analysis were used to investigate the orthologs and unique protein clusters in relation to morphotype. Results. WGS analysis showed high average nucleotide sequence identity (>98%), but genetic variants compared to the reference genome ranged from 1,433 to 27,025, and these differences did not correlate with morphotype. Among clinical isolates, smooth MABC clustered and were separated from the rough isolates. In both rough and smooth phenotypes, random genetic variations that could not separate the morphotypes were detected mainly in the nrps of the GPL loci. The major protein-cluster differences between smooth ( n =123) and rough ( n =22) clinical isolates were observed in key genes outside the GPL locus, such as fabH , pks15 , fadD29 , otaA , mabA , lysX and acdA , primarily involved in fatty acid biosynthesis and transporter systems. Repeated NO exposure, serving as a proxy for host-related stress, induced a rough phenotype by altering the acyl desaturase DesA1 protein, which is involved in fatty acid synthesis. Conclusion. By analysing clinical MABC isolates, we show that morphotype variation cannot be explained by the GPL locus only. Unique genes involved in fatty acid biosynthesis were identified, informing further investigation into mechanisms distinguishing smooth and rough morphotypes of clinical MABC isolates.