Lipid metabolic pathways determine phage infectivity in Mycobacterium abscessus
Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium with high intrinsic antibiotic resistance, requiring innovative therapeutics. During infection, smooth and rough colony morphotypes can coexist in the human body, participating in the pathogenesis. Despite its increasing clinical importance and phage therapy being a last resort treatment recently, the genetic basis of phage interaction in M. abscessus remains poorly understood. Previous work has focused largely on rough strains or the surrogate host Mycobacterium smegmatis. Here, we isolated novel phages that efficiently infect both morphotypes, allowing us to characterize phage-resistant mutants from paired smooth and rough clinical isolates to determine the genetic basis of the infectivity. Integrating whole-genome sequencing, transcriptomics, and phage susceptibility and adsorption assays, we deeply analyzed 30 phage-resistant variants and found that resistance trajectories were shaped primarily by host morphotype rather than by the selecting phage. We confirmed the TPP locus as a conserved determinant of phage infection in both morphotypes and identified previously undescribed hotspot mutations in furB and nrnA, together with a multi-gene deletion, in smooth-derived resistant variants. Whereas the TPP locus and the multi-gene deletion represent stable genomic changes affecting lipid-associated loci, frameshift mutations in furB and nrnA, not previously linked to lipid metabolism, were accompanied by broad transcriptional rewiring of lipid-related genes. Resistance was mutation-dependent and consistently associated with impaired phage adsorption, indicating an early block in infection. Together, these findings show that phage recognition in M. abscessus is shaped by mycomembrane lipid architecture rather than a single dedicated receptor and uncover regulatory and metabolic pathways with implications for more durable phage-based therapies.