Virginia Via Research Day Book 2026

Graduate Student Research Biomedical

03 EXTRACELLULAR MEMBRANE VESICLES: NEW PLAYERS IN STAPHYLOCOCCUS AUREUS BIOFILM BIOLOGY

Jinger Lei, Jaogang Wang, PhD Corresponding author: jingerlei@vt.edu

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia

wasutilized to characterize MV composition. We also recovered MVs from in vivo biofilms using a surgical mesh implant infection model, and immunoblotting was used to confirm their S. aureus origin. To assess the immunological relevance of biofilm MVs, human macrophages THP-1 were treated with purified biofilm MVs, and pro-inflammatory cytokine production was measured. Results: Our data indicate that approximately 237 particles were generated per bacterial colony-forming units (CFU) in biofilms, with an average size of 92.9 nm. Proteomic analysis revealed that S. aureus biofilm derived MVs are compositionally distinct from those produced under planktonic conditions. When add-ed to S. aureus planktonic cultures, only biofilm-derived MVs, but not planktonic MVs, significantly en-hanced S. aureus biofilm formation,suggesting a functional role of MVs in biofilm development. Im-portantly, we also recovered MVs from in vivo biofilms using a surgical mesh implant infection model. Immunoblotting showed that in vivo MVs were reactive withantibodies against S. aureus antigens α-toxin, lipoprotein MntC, and LTA, indicating that the recovered MVs were

S.aureus in origin. To assess the immunological relevance of biofilm MVs, human macrophages were treated withpurified biofilm MVs. Exposure to these MVs led to significant production of pro-inflammatory cytokines, suggesting arole for MVs in innate immune activation during biofilm-associated infections. Conclusion: Together, our findings demonstrate that S. aureus MVs are generated from biofilms and that these MVs contribute to both biofilm formation and inflammation during infection. Our experimental approaches are broadly applicable to other Gram positive pathogens and may enable the identification of novel bi-omarkers for diagnosing or monitoring biofilm-associated infections.

Background: Staphylococcus aureus is a major cause of biofilm-related infections on medical implants, where bio-films protectbacteria from immune defense and antibiotic treatment. Biofilm matrix is composed of pol-ysaccharides, proteins,and extracellular DNA. S. aureus also produces membrane vesicles (MVs) during planktonic growth, which have beenshown to play a role in the pathogenesis of S. aureus infections. However, whether MVs are produced in S. aureusbiofilms and contribute to biofilm formation is un-known. Objectives: We aimed to determine whether S. aureus produces MVs within biofilms and whether these MVs con-tribute to biofilm development and host immune activation. Methods: We cultured S. aureus biofilms in a drip-flow bioreactor and harvested biofilm matrix for MV purification with OptiPrep density gradient ultracentrifugation. The purified MVs were visualized by transmission electron microscopy (TEM), and their size and concentration quantified by nanoparticle tracking analy-sis (NTA). Proteomic analysis

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119 2026 Research Recognition Day