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

Abstract 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.