Ag85A-Dependent Surface Lipid Remodeling Impairs Adhesion and Infection of Therapeutic Mycobacteriophage

Patients infected with mycobacteria, particularly Mycobacterium abscessus, frequently experience antibiotic treatment failure. Phage therapy, used under compassionate care, offers a promising alternative and has already demonstrated clinical efficacy. However, phage-bacteria interactions and their coevolution remain critical factors that can compromise therapeutic success. Trehalose polyphleates (TPPs), complex lipids of the outer membrane, play a key role in the adhesion of the therapeutic mycobacteriophage BPsΔ33HTH_HRM10, and loss of TPP synthesis confers bacterial resistance to the phage. Herein, we found that deletion of the mycolyltransferase-encoding gene MSMEG_6398, but not of its homologues MSMEG_6396 or MSMEG_6399, all of which encode members of the Ag85 complex, increased resistance to BPsΔ33HTH_HRM10 on solid and liquid media. Alterations in the mycomembrane, the first barrier encountered by phages, were revealed by lipid profiling, while adhesion assays and flow cytometry indicated a defect at the binding stage. This effect was related to the accumulation of trehalose dimycolate (TDM) in the MSMEG_6398 mutant. The role of TDM in susceptibility/resistance to BPsΔ33HTH_HRM10 was then demonstrated with a knockdown mutant of the arabinosyltransferase AftB involved in a terminal step of cell wall arabinan biosynthesis associated with TDM accumulation and further validated using ethambutol, a drug that induces TDM accumulation. Collectively, these findings suggest that TPPs may be masked by excess trehalose-bound lipids, reducing phage adsorption and preventing subsequent phage infection.