Beyond Ribosomal Mutations: Identification of MPN_080 as a Novel ATPase-Dependent Determinant of Macrolide Resistance in <em>Mycoplasma pneumoniae</em>

Mycoplasma pneumoniae is a significant pathogen responsible for community-acquired respiratory infections in children and adolescents, with the rising prevalence of macrolide-resistant M. pneumoniae (MRMP), particularly in Asia, presenting critical treatment challenges. Our previous study inferred that macrolide efflux pump may contribute to macrolide resistance in M. pneumoniae in addition to the common point mutations in 23S rRNA gene. This study aimed to define the specific pump and confirm its role. Through comparative genomic analysis, we identified a candidate gene, MPN_080, encoding an ABC transporter permease, which was further characterized using phylogenetic analysis, AlphaFold-based structural modeling, and biochemical assays. Overexpression of MPN_080 from an erythromycin-resistant isolate in the erythromycin-sensitive M129, and resulted in a significant increase in minimum inhibitory concentrations (MICs) from &lt;0.125 µg/mL to 1 µg/mL, while similar overexpression of MPN_080 derived from M129 did not affect MICs. Notably, this resistance mechanism operates independently of M. pneumoniae virulence factors, as evidenced by unaltered colonization capacity in NCI-H292 cells and consistent immune response patterns across both strains. Our findings establish MPN_080 as a novel determinant of macrolide resistance functioning through an ATPase-dependent mechanism, distinct from classical 23S rRNA mutations. These insights into non-classical resistance mechanisms may guide future diagnostic and therapeutic strategies against MRMP.