Mycoplasma genitalium extracellular vesicles and the P140 adhesin carboxyl region as high-fidelity antigens for improved serodiagnosis

Mycoplasma genitalium ( Mg ) is an important etiological agent of sexually transmitted infections that has emerged as a global health threat. Despite its clinical importance, accurate serodiagnosis remains challenging due to high cross-reactivity caused by non-specific proteins. To address this limitation, we optimized Mg growth in a serum-free minimal medium to characterize the biogenesis and diagnostic applications of Mg -secreted extracellular vesicles (EVs). We demonstrate that Mg generates EVs that retain immunodominant proteins, specifically the key adhesins P140 and P110. From an initial cohort of 1,608 women (12.5% Mg- positive), we selected a longitudinal sub-study population, including 24 women who were PCR-positive at enrollment and a control group of 14 individuals who remained Mg -negative across three visits, each spaced approximately 6 months apart. Comparative ELISA and immunoblotting using sera from Mg -infected and uninfected individuals revealed that the EV-associated antigens provide superior diagnostic discrimination over conventional total-cell lysates and lipid-associated membrane protein extracts. Notably, EV-associated antigens lack common non-specific proteins, such as immunoglobulin-binding protein, MG281. Functional characterization using full-length and truncated P140 variants in cell-based ELISAs with human cervical epithelial cells identified the C-terminal region (P140-C) as the primary host-cell-interacting domain. In addition, this specific region induces an epitope-restricted antibody response in infected individuals. Within the longitudinal clinical cohort, high anti-P140-C antibody titers correlated with a trend toward Mg clearance, suggesting potential prognostic utility despite lacking statistical significance. Collectively, these results establish Mg EVs as a high-fidelity antigenic source and identify P140-C as a promising candidate for the development of next-generation serological assays. IMPORTANCE Serodiagnosis of Mycoplasma genitalium (Mg) faces significant challenges, especially in the context of emerging antimicrobial resistance and asymptomatic infections. This study introduces Mg -derived extracellular vesicles (EVs) as a source of refined antigens for serodiagnosis. By isolating the P140 C-terminal region, we identified a highly specific marker that overcomes antigenic variation, reduces cross-reactivity, and offers a candidate antigen that may warrant evaluation as a marker of infection clearance. By leveraging bacterial EVs as a source of purified diagnostic antigen, our findings offer a framework with broader implications for other antigenically complex pathogens.