Novel lipid-associated loci determine phage-host interaction in Mycobacterium abscessus

Mycobacterium abscessu s is a rapidly growing nontuberculous mycobacterium with high intrinsic antibiotic resistance, highlighting the need for innovative therapeutic strategies. During infection, smooth and rough colony morphotypes can coexist within the human host and contribute to pathogenesis. Despite the growing clinical relevance of M. abscessus and renewed interest in phage therapy as a last-resort treatment, the genetic basis of phage-host interactions remains poorly understood. Previous work has focused largely on rough strains or the surrogate host Mycobacterium smegmatis , proposing trehalose polyphleate (TPP), a surface-exposed glycolipid, as a co-receptor facilitating phage attachment. Here, we describe additional M. abscessus genes that modulate this initial step of infection. We isolated and characterized, using our in-house bioinformatic pipeline Phage2Pham, novel therapeutic phages that efficiently infect both morphotypes, enabling an in-depth characterization of stable phage-resistant mutants derived from paired smooth and rough clinical isolates. By integrating whole-genome sequencing and transcriptomics with phage susceptibility and adsorption assays, we confirmed TPP as a conserved determinant of phage infection in both morphotypes and identified a large genomic region encompassing more than 10 genes, together with furB and nrnA , as novel determinants of phage infection in smooth-derived resistant variants. All mutations were consistently associated with impaired phage adsorption, supporting a role for the affected loci in phage attachment, while functional genomic analyses linked these newly identified mutational hotspots to bacterial lipid metabolism. Specifically, phage resistance was associated either with genomic changes affecting lipid-associated loci within a large genomic region, with a distinct glycopeptidolipid-associated profile, or with frameshift mutations in furB and nrnA , leading to a downstream transcriptional rewiring of lipid-related genes. With previous studies proposing the involvement of surface-associated molecules in mycophage infection, our results extend this concept by identifying previously undescribed genetic loci potentially modulating the membrane lipid composition. Although direct functional validation is still required to establish the causal contribution of these loci, such experiments are particularly challenging in clinical M. abscessus strains. Nevertheless, these findings provide a new genetic framework for understanding phage-host interactions in mycobacteria and uncover potential regulatory and metabolic pathways with implications for the development of more durable phage-based therapies.