[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-114] Engineering bacterial extracellular vesicles using genetically encoded gas vesicles for non-invasive ultrasound imaging

○Daisuke Shimura¹, Mayu Kimoto¹, Yosuke Tashiro¹ (1. Grad. Sch. Intgr. Sci. Tech., Shizuoka Univ. (Japan))

Keywords:

Bacterial extracellular vesicles (BEVs),gas vesicles (GVs),ultrasound-responsive nanostructures,non-invasive imaging

Bacterial extracellular vesicles (BEVs) are nanoscale particles (~100 nm in diameter) released by bacteria that mediate intercellular communication through the transport of proteins, lipids, and nucleic acids. BEVs are increasingly recognized as functional biological nanoparticles involved in host–microbe interactions, including immune modulation and disease progression. However, their in vivo dynamics remain poorly understood due to the lack of non-invasive imaging strategies, particularly for deep tissue environments.In this study, we present a synthetic biology framework for engineering BEVs as ultrasound-responsive nanostructures by introducing genetically encoded gas vesicles (GVs) as functional cargo. GVs are protein-based nanostructures that enclose gas and generate acoustic contrast, making them promising candidates for ultrasound imaging.To reconstruct GV formation in a heterologous system, we selected 16 genes involved in gas vesicle biosynthesis from the 19-gene GV cluster of Serratia sp. ATCC 39006, focusing on structural and assembly components rather than transcription factors. We constructed an expression plasmid with these genes placed downstream of the lac promoter and transformed it into Escherichia coli. We performed heterologous expressions using the constructed E. coli under IPTG-induced conditions and evaluated GV formation by microscopic observation. This resulted in the formation of GV-like structures, demonstrating that the core machinery required for GV assembly can be functionally reconstituted in a non-native host.To facilitate the incorporation of GVs into BEVs, we designed a strategy based on subcellular localization control. First, signal peptides predicted to direct periplasmic localization were selected for each GV structural protein based on bioinformatics analysis. These signal peptides were fused to GV constituent proteins to direct their localization to the periplasmic space, which is the site of BEV biogenesis in Gram-negative bacteria. As a result, GV formation in the periplasm was suggested, indicating that the intracellular positioning of GVs can be rationally controlled. Because BEVs originate from the outer membrane and periplasm, this design is expected to promote the incorporation of GVs into BEVs during vesicle formation.This study provides a conceptual framework for engineering BEVs through genetically encoded cargo loading. By selectively reconstructing the minimal set of GV biosynthetic genes required for structural assembly, this work demonstrates a design strategy for introducing complex protein nanostructures into bacterial vesicles. Although direct encapsulation of GVs within BEVs and their detectability by ultrasound remains to be demonstrated, this approach establishes a foundation for the rational design of functionalized BEVs for non-invasive imaging applications.