A synonymous mutation in <i>MSMEG_4729</i> occurs at a high frequency in spontaneous D29-resistant mutants of <i>Mycobacterium smegmatis</i>

Abstract Compassionate use of mycobacteriophage therapy highlights the promising potential of phage therapy as an alternative treatment option for antibiotic-resistant infections when conventional treatments fail. However, realizing the full potential of phage therapy requires addressing key challenges, including host immune responses, the limited arsenal of therapeutically-useful mycobacteriophages, and the emergence of phage resistance. Dissecting the mechanisms of phage resistance is critical for ensuring the effectiveness and sustainability of phage therapy. In this study, we demonstrate that exposure to the lytic mycobacteriophage D29 triggers diverse genetic changes in Mycobacterium smegmatis . A synonymous mutation in MSMEG_4729 arises frequently but is insufficient to confer D29 resistance on its own. Instead, we identified possible Lsr2-independent activation of the lipooligosaccharide (LOS) biosynthesis cluster in a D29-resistant mutant harboring this mutation. We have also detected the possible activity of MSMEG_3213, a type II methyltransferase associated with m 6 A modifications in M. smegmatis . Finally, we isolated defense escape mutants (DEMs) of D29 capable of overcoming resistance in a strain with the MSMEG_4729 synonymous mutation. This profiling of M. smegmatis ’s likely defensive arsenal against the therapeutically-useful mycobacteriophage D29 provides a roadmap for further investigations and rational engineering of next-generation mycobacteriophages to combat drug-resistant mycobacterial infections. Impact statement Interest in phage therapy has been gaining traction recently, which is largely due to the serious threat of antimicrobial resistance. However, the efficacy and sustainability of phage therapy is threatened by certain challenges, which includes the ever existent threat of phage resistance. In this study, we identified several likely factors involved in D29 interaction with the model mycobacterium M. smegmatis . These findings set a roadmap for future investigations that would guide rational phage engineering to expand the currently limited arsenal of therapeutically useful mycobacteriophages as well as improve the efficiency of existing ones.